856 results on '"DNA and Chromosomes"'
Search Results
2. A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins
- Author
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Etsuko Shibata and Anindya Dutta
- Subjects
DNA Replication ,0301 basic medicine ,Origin Recognition Complex ,Replication Origin ,DNA and Chromosomes ,Biology ,Origin of replication ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Minichromosome maintenance ,Tumor Cells, Cultured ,medicine ,Humans ,ORC1 ,Molecular Biology ,Mutation ,030102 biochemistry & molecular biology ,Chromatin binding ,Cell Cycle ,DNA replication ,Cell Biology ,Chromatin ,Cell biology ,030104 developmental biology ,Colonic Neoplasms ,Cancer cell ,Origin recognition complex - Abstract
The Origin Recognition Complex (ORC), composed of six subunits, ORC1-6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2-7 around DNA and initiate DNA replication. We had reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a significant decrease in the number of origins firing. We now report that human HCT116 colon cancer cells also survive with a mutation in the initiator ATG of the ORC5 gene that abolishes the expression of ORC5 protein. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from similar number of origins as wild type cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells rendering both ORC5 and ORC2 proteins undetectable in the same cells, and destabilizing the ORC1, ORC3 and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2-7 normally to chromatin and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2-7 to initiate DNA replication, or human cell-lines can sometimes recruit MCM2-7 to origins independent of ORC.
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- 2020
3. Lysine acetylation regulates the activity of nuclear Pif1
- Author
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Christopher W. Sausen, Lata Balakrishnan, Matthew L. Bochman, and Onyekachi E. Ononye
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0301 basic medicine ,Saccharomyces cerevisiae Proteins ,DNA repair ,Lysine ,Saccharomyces cerevisiae ,DNA and Chromosomes ,Biochemistry ,Histone Deacetylases ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Tandem Mass Spectrometry ,DNA, Fungal ,Molecular Biology ,Histone Acetyltransferases ,Cell Nucleus ,030102 biochemistry & molecular biology ,biology ,DNA Helicases ,DNA replication ,Helicase ,Acetylation ,RNA, Fungal ,Cell Biology ,Cell biology ,030104 developmental biology ,chemistry ,Acetyltransferase ,Mutagenesis, Site-Directed ,biology.protein ,DNA ,Mitochondrial DNA replication - Abstract
In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo. Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.
- Published
- 2020
4. The DUF328 family member YaaA is a DNA-binding protein with a novel fold
- Author
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Janani Prahlad, Yifeng Yuan, Valérie de Crécy-Lagard, Jiusheng Lin, Mark A. Wilson, Chou Wei Chang, Yilun Liu, and Dirk Iwata-Reuyl
- Subjects
DNA, Bacterial ,0301 basic medicine ,Protein Folding ,DNA Repair ,DNA repair ,DNA and Chromosomes ,Crystallography, X-Ray ,Biochemistry ,DNA-binding protein ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,law ,Escherichia coli ,Holliday junction ,Molecular Biology ,Gene ,030102 biochemistry & molecular biology ,Escherichia coli Proteins ,Binding protein ,Cell Biology ,DNA-Binding Proteins ,Oxidative Stress ,030104 developmental biology ,Regulon ,chemistry ,Recombinant DNA ,DNA - Abstract
DUF328 family proteins are present in many prokaryotes, however their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Co-fitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to double-stranded DNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nM. YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65 A resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix (HhH) DNA binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.
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- 2020
5. AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair
- Author
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Xiaoyun Yang, Shih-Hsun Chen, Chen Wu, Anup Kumar Singh, Xiuhua Liu, Hongzhi Li, Lily L. Yu, Rong Xie, and Xiaochun Yu
- Subjects
0301 basic medicine ,DNA Repair ,Glycoside Hydrolases ,DNA repair ,In silico ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Line, Tumor ,medicine ,Humans ,Doxorubicin ,Enzyme Inhibitors ,Molecular Biology ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Cell Biology ,In vitro ,Cell biology ,030104 developmental biology ,Enzyme ,chemistry ,ADP-ribosylation ,Camptothecin ,DNA ,DNA Damage ,medicine.drug - Abstract
The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC(50) of ∼2.41 μm in vitro. Moreover, hydrolysis of DNA damage–induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.
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- 2020
6. R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities
- Author
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Lata Balakrishnan, Yanhao Lai, Eduardo E. Laverde, Yuan Liu, Catherine H. Freudenreich, and Fenfei Leng
- Subjects
0301 basic medicine ,DNA Repair ,Flap Endonucleases ,DNA repair ,DNA polymerase ,DNA damage ,Flap structure-specific endonuclease 1 ,DNA and Chromosomes ,Biochemistry ,AP endonuclease ,03 medical and health sciences ,Trinucleotide Repeats ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Humans ,AP site ,Molecular Biology ,DNA Polymerase beta ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Cell Biology ,Base excision repair ,Cell biology ,030104 developmental biology ,biology.protein ,R-Loop Structures ,Trinucleotide repeat expansion - Abstract
Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.
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- 2020
7. Binding and allosteric transmission of histone H3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 plant homeodomain finger
- Author
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Meiling R. May, John T. Bettridge, and Stephen Desiderio
- Subjects
0301 basic medicine ,education ,Allosteric regulation ,DNA and Chromosomes ,Methylation ,Biochemistry ,Substrate Specificity ,Histones ,Recombinases ,Mice ,03 medical and health sciences ,Histone H3 ,Allosteric Regulation ,Histone methylation ,Recombinase ,Animals ,VDJ Recombinases ,Molecular Biology ,Phylogeny ,Homeodomain Proteins ,Binding Sites ,030102 biochemistry & molecular biology ,Chemistry ,Lysine ,hemic and immune systems ,Cell Biology ,Chromatin ,V(D)J Recombination ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,PHD finger ,Sharks ,H3K4me3 ,Homeobox ,Protein Binding - Abstract
V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.
- Published
- 2020
8. Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes
- Author
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Joann B. Sweasy, Rebecca Kaup, Johannes Hohlbein, Jamie B. Towle-Weicksel, Meike Kronenberg, Mattia Fontana, Carel Fijen, and Mariam M. Mahmoud
- Subjects
DNA Replication ,Models, Molecular ,0301 basic medicine ,Conformational change ,DNA Repair ,Protein Conformation ,substrate specificity ,DNA repair ,DNA polymerase ,Biophysics ,Eukaryotic DNA replication ,DNA and Chromosomes ,Crystallography, X-Ray ,base excision repair ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,conformational change ,Fluorescence Resonance Energy Transfer ,Humans ,DNA binding ,BioNanoTechnology ,single-molecule analysis ,Molecular Biology ,DNA Polymerase beta ,030102 biochemistry & molecular biology ,biology ,Nucleotides ,Chemistry ,DNA ,Cell Biology ,Base excision repair ,Single-molecule FRET ,DNA Polymerase I ,DNA-Binding Proteins ,Kinetics ,Biofysica ,genome integrity ,030104 developmental biology ,fluorescence resonance energy transfer (FRET) ,biology.protein ,Nucleic Acid Conformation ,DNA construct - Abstract
Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.
- Published
- 2020
9. Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae
- Author
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Katrin Paeschke, Jozef Nosek, Lukáš Trantírek, Katarína Juríková, Mona Hajikazemi, Lubomir Tomaska, Katarina Prochazkova, and Martin Gajarsky
- Subjects
0301 basic medicine ,Telomerase ,Saccharomyces cerevisiae Proteins ,DNA damage ,Telomere-Binding Proteins ,Saccharomyces cerevisiae ,Oligonucleotides ,DNA, Single-Stranded ,Electrophoretic Mobility Shift Assay ,G-hairpin ,DNA and Chromosomes ,telomerase ,G-quadruplex ,Antiparallel (biochemistry) ,Biochemistry ,03 medical and health sciences ,folding kinetics ,Molecular Biology ,telomere ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Oligonucleotide ,Telomere Homeostasis ,DNA ,Cell Biology ,biology.organism_classification ,Telomere ,DNA-Binding Proteins ,G-Quadruplexes ,Kinetics ,030104 developmental biology ,Eukaryotic chromosome fine structure ,Cdc13 ,Biophysics ,Nucleic Acid Conformation ,cell cycle - Abstract
The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.
- Published
- 2020
10. Two components of DNA replication-dependent LexA cleavage
- Author
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Kamila K. Myka and Kenneth J. Marians
- Subjects
DNA Replication ,DNA, Bacterial ,0301 basic medicine ,DNA damage ,DNA repair ,DNA polymerase ,viruses ,DNA, Single-Stranded ,Pyrimidine dimer ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,Bacterial Proteins ,Escherichia coli ,SOS response ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Escherichia coli Proteins ,Serine Endopeptidases ,DNA replication ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,Cell biology ,DNA-Binding Proteins ,Rec A Recombinases ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Proteolysis ,biology.protein ,bacteria ,Replisome ,Repressor lexA - Abstract
Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA–RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.
- Published
- 2020
11. Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock
- Author
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Joseph J. Loparo, Alfredo J. Hernandez, Seungwoo Chang, Seung-Joo Lee, Charles C. Richardson, and Jaehun A. Lee
- Subjects
DNA Replication ,0301 basic medicine ,DNA polymerase ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Thioredoxins ,Protein Domains ,Bacteriophage T7 ,Escherichia coli ,Molecular Biology ,Polymerase ,Klenow fragment ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Escherichia coli Proteins ,DNA replication ,T7 DNA polymerase ,Cell Biology ,Processivity ,Molecular biology ,030104 developmental biology ,DNA, Viral ,biology.protein ,DNA polymerase I ,DNA - Abstract
Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.
- Published
- 2020
12. Ada protein– and sequence context–dependent mutagenesis of alkyl phosphotriester lesions in Escherichia coli cells
- Author
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Jiabin Wu, Jun Yuan, Yinsheng Wang, and Nathan E. Price
- Subjects
DNA Replication ,DNA, Bacterial ,0301 basic medicine ,Alkylation ,DNA polymerase ,DNA repair ,DNA damage ,DNA and Chromosomes ,medicine.disease_cause ,Biochemistry ,O(6)-Methylguanine-DNA Methyltransferase ,03 medical and health sciences ,chemistry.chemical_compound ,Escherichia coli ,medicine ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Escherichia coli Proteins ,Mutagenesis ,DNA replication ,Cell Biology ,Molecular biology ,DNA Alkylation ,030104 developmental biology ,chemistry ,biology.protein ,Gene Deletion ,DNA ,DNA Damage ,Transcription Factors - Abstract
Alkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- and n-butyl (nBu)-PTEs in two diastereomeric configurations (S(p) and R(p)) at six different flanking dinucleotide sites, i.e. XT and TX (X = A, C, or G), and assessed how these lesions impact DNA replication in Escherichia coli cells. When single-stranded vectors contained an S(p)-Me-PTE in the sequence contexts of 5′-AT-3′, 5′-CT-3′, or 5′-GT-3′, DNA replication was highly efficient and the replication products for all three sequence contexts contained 85–90% AT and 5–10% TG. Thus, the replication outcome was largely independent of the identity of the 5′ nucleotide adjacent to an S(p)-Me-PTE. Furthermore, replication across these lesions was not dependent on the activities of DNA polymerases II, IV, or V; Ada, a protein involved in adaptive response and repair of S(p)-Me-PTE in E. coli, however, was essential for the generation of the mutagenic products. Additionally, the R(p) diastereomer of Me-PTEs at XT sites and both diastereomers of Me-PTEs at TX sites exhibited error-free replication bypass. Moreover, S(p)-nBu-PTEs at XT sites did not strongly impede DNA replication, and other nBu-PTEs displayed moderate blockage effects, with none of them being mutagenic. Taken together, these findings provide in-depth understanding of how alkyl-PTE lesions are recognized by the DNA replication machinery in prokaryotic cells and reveal that Ada contributes to mutagenesis of S(p)-Me-PTEs in E. coli.
- Published
- 2020
13. Genome-wide single-nucleotide resolution of oxaliplatin–DNA adduct repair in drug-sensitive and -resistant colorectal cancer cell lines
- Author
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Christopher P. Selby, Yanyan Yang, Courtney M. Vaughn, Aziz Sancar, and David S. Hsu
- Subjects
0301 basic medicine ,Programmed cell death ,DNA Repair ,DNA damage ,DNA repair ,Drug resistance ,DNA and Chromosomes ,Biochemistry ,DNA Adducts ,03 medical and health sciences ,chemistry.chemical_compound ,DNA adduct ,medicine ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,DNA, Neoplasm ,Cell Biology ,HCT116 Cells ,Oxaliplatin ,030104 developmental biology ,chemistry ,Drug Resistance, Neoplasm ,Cancer research ,Colorectal Neoplasms ,DNA ,DNA Damage ,Nucleotide excision repair ,medicine.drug - Abstract
Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic, and approximately half of cancer patients have tumors that are either intrinsically resistant or develop resistance. Previous studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. Here, we aimed to better define the roles of nucleotide excision repair and DNA damage in platinum chemotherapy resistance by profiling DNA damage and repair efficiency in seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed DNA repair indirectly as toxicity and directly measured bulky adduct formation and removal from the genome by slot blot and repair capacity in an excision assay, and used excision repair sequencing (XR-seq) to map repair events genome-wide at single-nucleotide resolution. Using this combinatorial approach and proxies for oxaliplatin–DNA damage, we observed no significant differences in repair efficiency that could explain the relative sensitivities and chemotherapy resistances of these cell lines. In contrast, the levels of oxaliplatin-induced DNA damage were significantly lower in the resistant cells, indicating that decreased damage formation, rather than increased damage repair, is a major determinant of oxaliplatin resistance in these cell lines. XR-seq–based analysis of gene expression revealed up-regulation of membrane transport pathways in the resistant cells, and these pathways may contribute to resistance. In conclusion, additional research is needed to characterize the factors mitigating cellular DNA damage formation by platinum compounds.
- Published
- 2020
14. Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells
- Author
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Louise Prakash, Jung Hoon Yoon, Satya Prakash, and Robert E. Johnson
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DNA Replication ,0301 basic medicine ,Adenosine ,DNA Repair ,DNA damage ,DNA polymerase ,DNA Polymerase Theta ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,Catalytic Domain ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Conserved Sequence ,Polymerase ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Mutagenesis ,DNA replication ,Active site ,Cell Biology ,Cell biology ,030104 developmental biology ,biology.protein ,Error-free translesion synthesis ,DNA Damage - Abstract
The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N(6)-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.
- Published
- 2020
15. Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase
- Author
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Parminder Kaur, Matthew J. Longley, Hong Wang, Hai Pan, William C. Copeland, Preston Countryman, and Wendy Wang
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0301 basic medicine ,Mitochondrial DNA ,Base pair ,Mitochondrial disease ,DNA and Chromosomes ,Microscopy, Atomic Force ,Biochemistry ,DNA-binding protein ,Mitochondrial Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,DNA Helicases ,DNA replication ,Helicase ,DNA ,Cell Biology ,Processivity ,medicine.disease ,Recombinant Proteins ,Mitochondria ,Cell biology ,030104 developmental biology ,chemistry ,biology.protein ,Nucleic Acid Conformation ,Protein Multimerization ,Protein Binding - Abstract
Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.
- Published
- 2020
16. The roles of polymerases ν and θ in replicative bypass of O6- and N2-alkyl-2′-deoxyguanosine lesions in human cells
- Author
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Pengcheng Wang, Hua Du, Jun Wu, Yinsheng Wang, and Xiaomei He
- Subjects
0301 basic medicine ,Alkylation ,DNA Repair ,DNA damage ,DNA polymerase ,Guanine ,viruses ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,medicine ,Humans ,Molecular Biology ,Chromatography, High Pressure Liquid ,Polymerase ,Mutation ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Mutagenesis ,DNA replication ,Deoxyguanosine ,Cell Biology ,Molecular biology ,HEK293 Cells ,030104 developmental biology ,biology.protein ,DNA - Abstract
Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O(6)-alkyl-2′-deoxyguanosine (O(6)-alkyl-dG) and minor-groove N(2)-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O(6)-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O(6)-alkyl-dG lesions. O(6)-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O(6)-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O(6)-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O(6)-nBu-dG and O(6)-pyridyloxobutyl-dG. Replication across the two N(2)-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.
- Published
- 2020
17. Translesion synthesis DNA polymerases η, ι, and ν promote mutagenic replication through the anticancer nucleoside cytarabine
- Author
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Louise Prakash, Jayati Roy Choudhury, Jung Hoon Yoon, and Satya Prakash
- Subjects
DNA Replication ,0301 basic medicine ,Antimetabolites, Antineoplastic ,DNA polymerase ,DNA repair ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Humans ,Molecular Biology ,Polymerase ,030102 biochemistry & molecular biology ,biology ,DNA synthesis ,Chemistry ,Mutagenesis ,Cytarabine ,DNA replication ,Cell Biology ,Fibroblasts ,carbohydrates (lipids) ,Leukemia, Myeloid, Acute ,030104 developmental biology ,biology.protein ,Cancer research ,Nucleic Acid Conformation ,DNA ,medicine.drug - Abstract
Cytarabine (AraC) is the mainstay for the treatment of acute myeloid leukemia. Although complete remission is observed in a large proportion of patients, relapse occurs in almost all the cases. The chemotherapeutic action of AraC derives from its ability to inhibit DNA synthesis by the replicative polymerases (Pols); the replicative Pols can insert AraCTP at the 3′ terminus of the nascent DNA strand, but they are blocked at extending synthesis from AraC. By extending synthesis from the 3′-terminal AraC and by replicating through AraC that becomes incorporated into DNA, translesion synthesis (TLS) DNA Pols could reduce the effectiveness of AraC in chemotherapy. Here we identify the TLS Pols required for replicating through the AraC templating residue and determine their error-proneness. We provide evidence that TLS makes a consequential contribution to the replication of AraC-damaged DNA; that TLS through AraC is conducted by three different pathways dependent upon Polη, Polι, and Polν, respectively; and that TLS by all these Pols incurs considerable mutagenesis. The prominent role of TLS in promoting proficient and mutagenic replication through AraC suggests that TLS inhibition in acute myeloid leukemia patients would increase the effectiveness of AraC chemotherapy; and by reducing mutation formation, TLS inhibition may dampen the emergence of drug-resistant tumors and thereby the high incidence of relapse in AraC-treated patients.
- Published
- 2019
18. Protein phosphatase 2A controls ongoing DNA replication by binding to and regulating cell division cycle 45 (CDC45)
- Author
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Goutham Narla, Abbey L. Perl, Caitlin M. O’Connor, Junran Zhang, Youwei Zhang, Franklin Mayca Pozo, and Pengyan Fa
- Subjects
DNA Replication ,0301 basic medicine ,Cell cycle checkpoint ,Cell Cycle Proteins ,Ataxia Telangiectasia Mutated Proteins ,macromolecular substances ,DNA and Chromosomes ,Biology ,environment and public health ,Biochemistry ,S Phase ,Mice ,03 medical and health sciences ,Cell Line, Tumor ,Animals ,Protein–DNA interaction ,Protein Phosphatase 2 ,Molecular Biology ,Gene ,030102 biochemistry & molecular biology ,DNA replication ,Cell Biology ,Protein phosphatase 2 ,Cell cycle ,Cell biology ,Enzyme Activation ,enzymes and coenzymes (carbohydrates) ,Cell Transformation, Neoplastic ,030104 developmental biology ,embryonic structures ,Replisome ,Female ,Homologous recombination ,DNA Damage ,Protein Binding - Abstract
Genomic replication is a highly regulated process and represents both a potential benefit and liability to rapidly dividing cells; however, the precise post-translational mechanisms regulating genomic replication are incompletely understood. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that regulates a diverse array of cellular processes. Here, utilizing both a gain-of-function chemical biology approach and loss-of-function genetic approaches to modulate PP2A activity, we found that PP2A regulates DNA replication. We demonstrate that increased PP2A activity can interrupt ongoing DNA replication, resulting in a prolonged S phase. The impaired replication resulted in a collapse of replication forks, inducing dsDNA breaks, homologous recombination, and a PP2A-dependent replication stress response. Additionally, we show that during replication, PP2A exists in complex with cell division cycle 45 (CDC45) and that increased PP2A activity caused dissociation of CDC45 and polymerase α from the replisome. Furthermore, we found that individuals harboring mutations in the PP2A Aα gene have a higher fraction of genomic alterations, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. These results reveal a new function for PP2A in regulating ongoing DNA replication and a potential role for PP2A in the intra-S-phase checkpoint.
- Published
- 2019
19. 5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells
- Author
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Nicholas E. Geacintov, Suse Broyde, Shaofei Ji, Marina Kolbanovskiy, Natalia Y. Tretyakova, Maram M. Essawy, Konstantin Kropachev, Iwen Fu, and Daeyoon Park
- Subjects
DNA Replication ,0301 basic medicine ,DNA Repair ,Transcription, Genetic ,DNA damage ,RNA polymerase II ,DNA and Chromosomes ,Biochemistry ,Cell Line ,Cytosine ,03 medical and health sciences ,chemistry.chemical_compound ,Transcription (biology) ,Gene expression ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,Regulation of gene expression ,030102 biochemistry & molecular biology ,biology ,DNA ,Cell Biology ,Cell biology ,Cross-Linking Reagents ,HEK293 Cells ,030104 developmental biology ,Histone ,chemistry ,biology.protein ,Peptides ,HeLa Cells ,Nucleotide excision repair - Abstract
5-Formylcytosine (5fC) is an endogenous epigenetic DNA mark introduced via enzymatic oxidation of 5-methyl-dC in DNA. We and others recently reported that 5fC can form reversible DNA–protein conjugates with histone proteins, likely contributing to regulation of nucleosomal organization and gene expression. The protein component of DNA–protein cross-links can be proteolytically degraded, resulting in smaller DNA–peptide cross-links. Unlike full-size DNA–protein cross-links that completely block replication and transcription, DNA–peptide cross-links can be bypassed by DNA and RNA polymerases and can potentially be repaired via the nucleotide excision repair (NER) pathway. In the present work, we constructed plasmid molecules containing reductively stabilized, site-specific 5fC–polypeptide lesions and employed a quantitative MS-based assay to assess their effects on transcription in cells. Our results revealed that the presence of DNA–peptide cross-link significantly inhibits transcription in human HEK293T cells but does not induce transcription errors. Furthermore, transcription efficiency was similar in WT and NER-deficient human cell lines, suggesting that the 5fC–polypeptide lesion is a weak substrate for NER. This finding was confirmed by in vitro NER assays in cell-free extracts from human HeLa cells, suggesting that another mechanism is required for 5fC–polypeptide lesion removal. In summary, our findings indicate that 5fC-mediated DNA–peptide cross-links dramatically reduce transcription efficiency, are poor NER substrates, and do not cause transcription errors.
- Published
- 2019
20. The finger loop of the SRA domain in the E3 ligase UHRF1 is a regulator of ubiquitin targeting and is required for the maintenance of DNA methylation
- Author
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Robert M. Vaughan, Scott B. Rothbart, and Bradley M. Dickson
- Subjects
0301 basic medicine ,Ubiquitin-Protein Ligases ,DNA-binding protein ,DNA and Chromosomes ,Biochemistry ,DNA methyltransferase ,Phosphates ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Ubiquitin ,Ring finger ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,DNA methylation ,epigenetics ,030102 biochemistry & molecular biology ,biology ,DNA ,Cell Biology ,HCT116 Cells ,molecular dynamics ,allosteric regulation ,Cell biology ,Ubiquitin ligase ,HEK293 Cells ,030104 developmental biology ,Histone ,medicine.anatomical_structure ,E3 ubiquitin ligase ,chemistry ,ubiquitin-like with PHD and RING finger domains 1 (UHRF1) ,SRA domain ,CCAAT-Enhancer-Binding Proteins ,biology.protein ,string method in collective variables - Abstract
The Su(var)3–9, enhancer of zeste, and trithorax (SET) and really interesting new gene (RING) finger–associated (SRA) protein domain is conserved across bacteria and eukaryota and coordinates extrahelical or “flipped” DNA bases. A functional SRA domain is required for ubiquitin-like with PHD and RING finger domains 1 (UHRF1) E3 ubiquitin ligase activity toward histone H3, a mechanism for recruiting the DNA methylation maintenance enzyme DNA methyltransferase 1 (DNMT1). The SRA domain supports UHRF1 oncogenic activity in colon cancer cells, highlighting that UHRF1 SRA antagonism could be a cancer therapeutic strategy. Here we used molecular dynamics simulations, DNA binding assays, in vitro ubiquitination reactions, and DNA methylation analysis to identify the SRA finger loop as a regulator of UHRF1 ubiquitin targeting and DNA methylation maintenance. A chimeric UHRF1 (finger swap) with diminished E3 ligase activity toward nucleosomal histones, despite tighter binding to unmodified or asymmetric or symmetrically methylated DNA, uncouples DNA affinity from regulation of E3 ligase activity. Our model suggests that SRA domains sample DNA bases through flipping in the presence or absence of a cytosine modification and that specific interactions of the SRA finger loop with DNA are required for downstream host protein function. Our findings provide insight into allosteric regulation of UHRF1 E3 ligase activity, suggesting that UHRF1's SRA finger loop regulates its conformation and function.
- Published
- 2019
21. The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF
- Author
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Andrew L. Wood, Anne E. Corcoran, Changfeng Chen, Colette Johnston, Marjorie A. Oettinger, John W. Morris, Yanqun Wang, Ruslan I. Sadreyev, Yuka Namiki, Adam G. W. Matthews, Katrina B. Morshead, and David N. Ciccone
- Subjects
0301 basic medicine ,CCCTC-Binding Factor ,DNA recombination ,Immunoglobulin Variable Region ,antigen receptor ,Locus (genetics) ,Adaptive Immunity ,Regulatory Sequences, Nucleic Acid ,DNA and Chromosomes ,Biology ,Biochemistry ,Mice ,03 medical and health sciences ,Transcription (biology) ,Recombinase ,Animals ,Humans ,Recombination signal sequences ,Nucleotide Motifs ,DNA binding ,CCTC-binding factor (CTCF) ,cellular immune response ,chromatin regulation ,Molecular Biology ,Transcription factor ,Gene Rearrangement ,Mice, Knockout ,Genetics ,Binding Sites ,030102 biochemistry & molecular biology ,Cell Biology ,Chromatin ,DNA-Binding Proteins ,Repressor Proteins ,030104 developmental biology ,CTCF ,NIH 3T3 Cells ,V(D)J ,chromatin immunoprecipitation (ChIP) ,PAX5 ,Immunoglobulin Heavy Chains ,K562 Cells - Abstract
Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP–chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.
- Published
- 2019
22. DNA duplex recognition activates Exo1 nuclease activity
- Author
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Hengyao Niu, Jiangchuan Shen, and Yuxi Li
- Subjects
0301 basic medicine ,DNA repair ,DNA, Single-Stranded ,DNA and Chromosomes ,Biochemistry ,Catalysis ,Substrate Specificity ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,law ,Replication Protein A ,Molecular Biology ,Replication protein A ,Nuclease ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Oligonucleotide ,Chemistry ,Lysine ,Cell Biology ,enzymes and coenzymes (carbohydrates) ,DNA Repair Enzymes ,Exodeoxyribonucleases ,030104 developmental biology ,Duplex (building) ,Mutation ,biology.protein ,Recombinant DNA ,Homologous recombination ,DNA - Abstract
Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability. The biochemical attributes of Exo1 have been extensively characterized via conventional assays. However, the key step governing its activation remains elusive. Extending the previous finding that Exo1 can digest a randomly selected single-stranded DNA (ssDNA) but not a poly(dT) oligonucleotide and using purified recombinant Exo1 and nuclease and electrophoretic mobility shift assays, here we determined that DNA hairpins with a stem size of 4 bp or longer are able to activate Exo1-mediated digestion of ssDNA. We further provide evidence suggesting that Exo1 uses an evolutionarily conserved residue, Lys(185). This residue interacted with the phosphate group bridging the third and fourth nucleotide on the digestion strand of the substrate DNA for duplex recognition, critical for Exo1 activation on not only ssDNA but also dsDNA. Additionally, the defect of an exo1-K185A mutant in duplex digestion was partially rescued by longer overhanging DNA. However, we noted that the enhanced Exo1 nuclease activity by longer overhanging DNA is largely eliminated by replication protein A (RPA), likely because of the previously reported RPA activity that strips Exo1 off the ssDNA. We conclude that duplex DNA contact by Exo1 is a general mechanism that controls its activation and that this mechanism is particularly important for digestion of duplex DNA whose nascent ssDNA is bound by RPA.
- Published
- 2019
23. Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells
- Author
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Claudia M. Nicolae, George Lucian Moldovan, Jenna Thomforde, Zhongtao Zhang, Natalia Y. Tretyakova, Shaofei Ji, Marietta Y.W.T. Lee, Spandana Naldiga, and Ashis K. Basu
- Subjects
DNA Replication ,0301 basic medicine ,DNA damage ,DNA polymerase ,DNA repair ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,Cytosine ,DNA Adducts ,Gene Knockout Techniques ,03 medical and health sciences ,chemistry.chemical_compound ,Plasmid ,Humans ,Molecular Biology ,reproductive and urinary physiology ,Polymerase ,DNA Polymerase III ,DNA Primers ,030102 biochemistry & molecular biology ,biology ,DNA replication ,DNA ,DNA Polymerase II ,Cell Biology ,Molecular biology ,Chromatin ,HEK293 Cells ,030104 developmental biology ,chemistry ,Mutation ,embryonic structures ,biology.protein ,Peptides - Abstract
DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.
- Published
- 2019
24. Stella protein facilitates DNA demethylation by disrupting the chromatin association of the RING finger–type E3 ubiquitin ligase UHRF1
- Author
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Ting Zhou, Bing Zhu, Wenlong Du, Hailin Wang, Zhuqiang Zhang, Baodong Liu, Yingfeng Li, Qiang Dong, and Rui-Ming Xu
- Subjects
0301 basic medicine ,Chromosomal Proteins, Non-Histone ,Ubiquitin-Protein Ligases ,Active Transport, Cell Nucleus ,DNA and Chromosomes ,Biochemistry ,DNA methyltransferase ,Histones ,03 medical and health sciences ,Histone H3 ,Protein Domains ,Ring finger ,medicine ,Humans ,Nuclear export signal ,Molecular Biology ,Demethylation ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Cell Biology ,Chromatin ,Ubiquitin ligase ,Cell biology ,DNA Demethylation ,HEK293 Cells ,030104 developmental biology ,medicine.anatomical_structure ,DNA demethylation ,Mutagenesis ,CCAAT-Enhancer-Binding Proteins ,biology.protein ,Protein Binding - Abstract
Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger–type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.
- Published
- 2019
25. Common motifs in ETAA1 and TOPBP1 required for ATR kinase activation
- Author
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Vaughn Thada and David Cortez
- Subjects
0301 basic medicine ,DNA damage ,DNA repair ,Amino Acid Motifs ,Ataxia Telangiectasia Mutated Proteins ,Serine threonine protein kinase ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,Protein Domains ,stomatognathic system ,Cell Line, Tumor ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,Kinase ,Activator (genetics) ,Chemistry ,Binding protein ,DNA replication ,Nuclear Proteins ,Cell Biology ,Cell cycle ,Cell biology ,DNA-Binding Proteins ,Enzyme Activation ,HEK293 Cells ,030104 developmental biology ,Antigens, Surface ,biological phenomena, cell phenomena, and immunity ,Carrier Proteins - Abstract
DNA damage response Ser/Thr kinases, including ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR), control cell cycle progression, DNA repair, and apoptosis. ATR is activated by ETAA1 activator of ATR kinase (ETAA1) or DNA topoisomerase II binding protein 1 (TOPBP1). Both ETAA1 and TOPBP1 contain experimentally defined ATR activation domains (AADs) that are mostly unstructured and have minimal sequence similarity. A tryptophan residue in both AADs is required for ATR activation, but the other features of these domains and the mechanism by which they activate ATR are unknown. In this study, using bioinformatic analyses, kinase assays, co-immunoprecipitation, and immunofluorescence measures of signaling, we more specifically defined the TOPBP1 and ETAA1 AADs and identified additional features of the AADs needed for ATR activation. We found that both ETAA1 and TOPBP1 contain a predicted coiled-coil motif that is required for ATR activation in vitro and in cells. Mutation of the predicted coiled coils does not alter AAD oligomerization but does impair binding of the AADs to ATR. These results suggest that TOPBP1 and ETAA1 activate ATR using similar motifs and mechanisms.
- Published
- 2019
26. A molecular model for self-assembly of the synaptonemal complex protein SYCE3
- Author
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Dunne, Orla M. and Davies, Owen R.
- Subjects
chromatin structure ,Models, Molecular ,Protein Conformation, alpha-Helical ,chromosomes ,molecular modeling ,Synaptonemal Complex ,small-angle X-ray scattering (SAXS) ,Cell Cycle Proteins ,SYCE3 ,DNA and Chromosomes ,protein self-assembly ,Crystallography, X-Ray ,Recombinant Proteins ,domain swap ,X-Ray Diffraction ,biophysics ,Scattering, Small Angle ,Mutagenesis, Site-Directed ,coiled-coil ,meiosis ,structural biology ,Humans ,Amino Acid Sequence ,protein structure ,Dimerization - Abstract
The synaptonemal complex (SC) is a supramolecular protein assembly that mediates homologous chromosome synapsis during meiosis. This zipper-like structure assembles in a continuous manner between homologous chromosome axes, enforcing a 100-nm separation along their entire length and providing the necessary three-dimensional framework for cross-over formation. The mammalian SC comprises eight components-synaptonemal complex protein 1-3 (SYCP1-3), synaptonemal complex central element protein 1-3 (SYCE1-3), testis-expressed 12 (TEX12), and six6 opposite strand transcript 1 (SIX6OS1)-arranged in transverse and longitudinal structures. These largely α-helical, coiled-coil proteins undergo heterotypic interactions, coupled with recursive self-assembly of SYCP1, SYCE2-TEX12, and SYCP2-SYCP3, to achieve the vast supramolecular SC structure. Here, we report a novel self-assembly mechanism of the SC central element component SYCE3, identified through multi-angle light scattering and small-angle X-ray scattering (SAXS) experiments. These analyses revealed that SYCE3 adopts a dimeric four-helical bundle structure that acts as the building block for concentration-dependent self-assembly into a series of discrete higher-order oligomers. We observed that this is achieved through staggered lateral interactions between self-assembly surfaces of SYCE3 dimers and through end-on interactions that likely occur through intermolecular domain swapping between dimer folds. These mechanisms are combined to achieve potentially limitless SYCE3 assembly, particularly favoring formation of dodecamers of three laterally associated end-on tetramers. Our findings extend the family of self-assembling proteins within the SC and reveal additional means for structural stabilization of the SC central element.
- Published
- 2019
27. Loss of the tumor suppressor BIN1 enables ATM Ser/Thr kinase activation by the nuclear protein E2F1 and renders cancer cells resistant to cisplatin
- Author
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Slovénie Pyndiah, Daitoku Sakamuro, Amy L. Abdulovic-Cui, Erica K. Cassimere, Watson P. Folk, Joanna C. Johnson, Alpana Kumari, and Tetsushi Iwasaki
- Subjects
0301 basic medicine ,DNA Repair ,Transcription, Genetic ,Tumor suppressor gene ,Cell Cycle Proteins ,Ataxia Telangiectasia Mutated Proteins ,DNA and Chromosomes ,Biochemistry ,Histones ,03 medical and health sciences ,Cell Line, Tumor ,Neoplasms ,medicine ,Humans ,E2F1 ,DNA Breaks, Double-Stranded ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,E2F2 ,Cisplatin ,MRE11 Homologue Protein ,030102 biochemistry & molecular biology ,Chemistry ,Tumor Suppressor Proteins ,Nuclear Proteins ,DNA, Neoplasm ,Cell Biology ,G2-M DNA damage checkpoint ,medicine.disease ,Acid Anhydride Hydrolases ,Cell biology ,MDC1 ,DNA-Binding Proteins ,DNA Repair Enzymes ,030104 developmental biology ,MRN complex ,Drug Resistance, Neoplasm ,Ataxia-telangiectasia ,biological phenomena, cell phenomena, and immunity ,E2F1 Transcription Factor ,Signal Transduction ,medicine.drug - Abstract
The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia–mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.
- Published
- 2019
28. Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen
- Author
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Stephen J. Benkovic, Anthony M. Pedley, Mahesh Aitha, and Mark Hedglin
- Subjects
0301 basic medicine ,DNA polymerase ,Ubiquitin-Protein Ligases ,DNA, Single-Stranded ,DNA and Chromosomes ,complex mixtures ,Biochemistry ,03 medical and health sciences ,Proliferating Cell Nuclear Antigen ,Replication Protein A ,Humans ,Monoubiquitination ,Protein Interaction Maps ,Molecular Biology ,Replication protein A ,DNA clamp ,Nucleoplasm ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Ubiquitination ,DNA replication ,Cell Biology ,Proliferating cell nuclear antigen ,Cell biology ,Chromatin ,DNA-Binding Proteins ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,Ubiquitin-Conjugating Enzymes ,biology.protein - Abstract
DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm. However, it is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Here, we developed an in vitro assay to quantitatively monitor PCNA monoubiquitination under in vivo scenarios. Results from extensive experiments revealed that RPA regulates Rad6/Rad18 activity in an ssDNA-dependent manner. We found that “DNA-free” RPA inhibits monoubiquitination of free PCNA by directly interacting with Rad18. This interaction is promoted under native conditions when there is an overabundance of free RPA in the nucleoplasm where Rad6/Rad18 and a significant fraction of PCNA reside. During DNA replication stress, RPA binds the ssDNA exposed downstream of stalled primer/template (P/T) junctions, releasing Rad6/Rad18. RPA restricted the resident PCNAs to the upstream duplex regions by physically blocking diffusion of PCNA along ssDNA, and this activity was required for efficient monoubiquitination of PCNA on DNA. Furthermore, upon binding ssDNA, RPA underwent a conformational change that increased its affinity for Rad18. Rad6/Rad18 complexed with ssDNA-bound RPA was active, and this interaction may selectively promote monoubiquitination of PCNA on long RPA-coated ssDNA.
- Published
- 2019
29. Function of a strand-separation pin element in the PriA DNA replication restart helicase
- Author
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James L. Keck, Maxime Leroux, Steven J. Sandler, and Tricia A. Windgassen
- Subjects
DNA Replication ,DNA, Bacterial ,0301 basic medicine ,Protein Conformation ,DNA repair ,Mutant ,DNA, Single-Stranded ,DNA and Chromosomes ,Crystallography, X-Ray ,Biochemistry ,Primosome ,03 medical and health sciences ,chemistry.chemical_compound ,Escherichia coli ,Protein–DNA interaction ,Amino Acid Sequence ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Escherichia coli Proteins ,DNA Helicases ,DNA replication ,Helicase ,DNA ,Cell Biology ,Primosome complex ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,biology.protein ,Nucleic Acid Conformation - Abstract
DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating “pin” structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein–DNA cross-linking, we localized the putative pin (a β-hairpin within a zinc-binding domain in PriA) near the ssDNA–dsDNA junction of the lagging strand in a PriA–DNA replication fork complex. Removal of residues at the tip of the β-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA β-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted β-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro. These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.
- Published
- 2019
30. Dynamic interactions of the homologous pairing 2 (Hop2)–meiotic nuclear divisions 1 (Mnd1) protein complex with meiotic presynaptic filaments in budding yeast
- Author
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Eric C. Greene, Youngho Kwon, Patrick Sung, and J. Brooks Crickard
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,DNA repair ,Saccharomyces cerevisiae ,RAD51 ,Cell Cycle Proteins ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,Meiosis ,Recombinase ,Protein–DNA interaction ,Protein Interaction Maps ,Homologous Recombination ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,fungi ,Cell Biology ,biology.organism_classification ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,DMC1 ,Homologous recombination ,Protein Binding - Abstract
Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2–Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2–Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51– and Dmc1–ssDNA in real time. We found that yeast Hop2–Mnd1 bound rapidly to Dmc1–ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2–Mnd1 with Rad51–ssDNA or RPA–ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2–Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR.
- Published
- 2019
31. Poly(ADP-ribose) polymerase 1 (PARP1) promotes oxidative stress–induced association of Cockayne syndrome group B protein with chromatin
- Author
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Robert J. Lake, Kostiantyn Dreval, Hua-Ying Fan, and Erica L. Boetefuer
- Subjects
musculoskeletal diseases ,0301 basic medicine ,CCCTC-Binding Factor ,congenital, hereditary, and neonatal diseases and abnormalities ,Ultraviolet Rays ,DNA repair ,Poly ADP ribose polymerase ,Poly (ADP-Ribose) Polymerase-1 ,DNA and Chromosomes ,Biochemistry ,Cockayne syndrome ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,medicine ,Transcriptional regulation ,Humans ,Poly-ADP-Ribose Binding Proteins ,Molecular Biology ,Polymerase ,biology ,Chemistry ,DNA Helicases ,Vitamin K 3 ,nutritional and metabolic diseases ,DNA ,Cell Biology ,medicine.disease ,Chromatin ,Cell biology ,Oxidative Stress ,DNA Repair Enzymes ,030104 developmental biology ,Histone ,Genetic Loci ,Mutation ,biology.protein ,030217 neurology & neurosurgery ,DNA Damage ,Protein Binding - Abstract
Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that relieves oxidative stress by regulating DNA repair and transcription. CSB is proposed to participate in base-excision repair (BER), the primary pathway for repairing oxidative DNA damage, but exactly how CSB participates in this process is unknown. It is also unclear whether CSB contributes to other repair pathways during oxidative stress. Here, using a patient-derived CS1AN-sv cell line, we examined how CSB is targeted to chromatin in response to menadione-induced oxidative stress, both globally and locus-specifically. We found that menadione-induced, global CSB–chromatin association does not require CSB's ATPase activity and is, therefore, mechanistically distinct from UV-induced CSB–chromatin association. Importantly, poly(ADP-ribose) polymerase 1 (PARP1) enhanced the kinetics of global menadione-induced CSB–chromatin association. We found that the major BER enzymes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), do not influence this association. Additionally, the level of γ-H2A histone family member X (γ-H2AX), a marker for dsDNA breaks, was not increased in menadione-treated cells. Therefore, our results support a model whereby PARP1 localizes to ssDNA breaks and recruits CSB to participate in DNA repair. Furthermore, this global CSB–chromatin association occurred independently of RNA polymerase II–mediated transcription elongation. However, unlike global CSB–chromatin association, both PARP1 knockdown and inhibition of transcription elongation interfered with menadione-induced CSB recruitment to specific genomic regions. This observation supports the hypothesis that CSB is also targeted to specific genomic loci to participate in transcriptional regulation in response to oxidative stress.
- Published
- 2018
32. Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences
- Author
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Yue Ma, Keisuke Iida, Hisao Masai, Yutaka Kanoh, Naoko Kakusho, Rino Fukatsu, and Kazuo Nagasawa
- Subjects
DNA Replication ,0301 basic medicine ,Molecular Sequence Data ,Telomere-Binding Proteins ,DNA Footprinting ,DNA, Single-Stranded ,DNA footprinting ,DNA and Chromosomes ,G-quadruplex ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Schizosaccharomyces ,DNA Replication Timing ,Nucleic acid structure ,DNA, Fungal ,Molecular Biology ,Nuclease ,Binding Sites ,Base Sequence ,biology ,DNA replication ,Cell Biology ,biology.organism_classification ,G-Quadruplexes ,030104 developmental biology ,chemistry ,Schizosaccharomyces pombe ,biology.protein ,Biophysics ,Nucleic Acid Conformation ,Schizosaccharomyces pombe Proteins ,DNA - Abstract
G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.
- Published
- 2018
33. Expression of Concern: Sirtuin 1-mediated deacetylation of XPA DNA repair protein enhances its interaction with ATR protein and promotes cAMP-induced DNA repair of UV damage
- Subjects
enzymes and coenzymes (carbohydrates) ,endocrine system ,biological phenomena, cell phenomena, and immunity ,DNA and Chromosomes ,Expressions of Concern - Abstract
Blunted melanocortin 1 receptor (MC1R) signaling promotes melanocyte genomic instability in part by attenuating cAMP-mediated DNA repair responses, particularly nucleotide excision repair (NER), which recognizes and clears mutagenic photodamage. cAMP-enhanced NER is mediated by interactions between the ataxia telangiectasia-mutated and Rad3-related (ATR) and xeroderma pigmentosum complementation group A (XPA) proteins. We now report a critical role for sirtuin 1 (SIRT1) in regulating ATR-mediated phosphorylation of XPA. SIRT1 deacetylates XPA at residues Lys-63, Lys-67, and Lys-215 to promote interactions with ATR. Mutant XPA containing acetylation mimetics at residues Lys-63, Lys-67, and Lys-215 exhibit blunted UV-dependent ATR–XPA interactions even in the presence of cAMP signals. ATR-mediated phosphorylation of XPA on Ser-196 enhances cAMP-mediated optimization of NER and is promoted by SIRT1-mediated deacetylation of XPA on Lys-63, Lys-67, and Lys-215. Interference with ATR-mediated XPA phosphorylation at Ser-196 by persistent acetylation of XPA at Lys-63, Lys-67, and Lys-215 delays repair of UV-induced DNA damage and attenuates cAMP-enhanced NER. Our study identifies a regulatory ATR–SIRT1–XPA axis in cAMP-mediated regulation melanocyte genomic stability, involving SIRT1-mediated deacetylation (Lys-63, Lys-67, and Lys-215) and ATR-dependent phosphorylation (Ser-196) post-translational modifications of the core NER factor XPA.
- Published
- 2020
34. An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3
- Author
-
Jessica L. Wojtaszek, Tuhin Haldar, Alyssa A. Rodriguez, Brandt F. Eichman, R. Scott Williams, Briana H. Greer, and Kent S. Gates
- Subjects
0301 basic medicine ,DNA Replication ,DNA damage ,DNA repair ,DNA, Single-Stranded ,DNA and Chromosomes ,Crystallography, X-Ray ,Biochemistry ,DNA-binding protein ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Animals ,Humans ,Protein–DNA interaction ,Amino Acid Sequence ,Molecular Biology ,N-Glycosyl Hydrolases ,chemistry.chemical_classification ,Zinc finger ,030102 biochemistry & molecular biology ,Zinc Fingers ,Cell Biology ,DNA ,Cell biology ,Protein Structure, Tertiary ,030104 developmental biology ,Enzyme ,chemistry ,DNA glycosylase ,Sequence Alignment ,Protein Binding - Abstract
The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF–ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF–ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF–ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF–ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.
- Published
- 2020
35. Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis
- Author
-
Melanie A. Sparks, Peter M. J. Burgers, and Roberto Galletto
- Subjects
0301 basic medicine ,DNA Replication ,Saccharomyces cerevisiae Proteins ,DNA polymerase ,Saccharomyces cerevisiae ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,Endonuclease ,chemistry.chemical_compound ,Acetyltransferases ,Replication Protein A ,Nucleosome ,Nick translation ,DNA, Fungal ,Molecular Biology ,DNA Polymerase III ,030102 biochemistry & molecular biology ,biology ,DNA synthesis ,Chemistry ,DNA replication ,DNA Helicases ,Helicase ,Membrane Proteins ,Cell Biology ,Cell biology ,030104 developmental biology ,biology.protein ,DNA - Abstract
Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication.
- Published
- 2020
36. Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities
- Author
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Jamie Baxter, Barbara E. Funnell, and William G. Waples
- Subjects
0301 basic medicine ,DNA, Bacterial ,Operator Regions, Genetic ,Mutation, Missense ,Repressor ,DNA Primase ,DNA and Chromosomes ,Biochemistry ,DNA-binding protein ,Plasmid maintenance ,03 medical and health sciences ,chemistry.chemical_compound ,Viral Proteins ,Plasmid ,Escherichia coli ,Protein–DNA interaction ,Bacteriophage P1 ,Molecular Biology ,030102 biochemistry & molecular biology ,Chemistry ,Circular bacterial chromosome ,Escherichia coli Proteins ,Cell Biology ,Bacterial nucleoid ,Chromosomes, Bacterial ,Cell biology ,030104 developmental biology ,Amino Acid Substitution ,DNA - Abstract
The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg(351), a residue previously predicted to participate in site-specific DNA binding. In vivo, the parA(R351A) allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a “super-repressor.” In vitro, ParA(R351A) binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.
- Published
- 2020
37. Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex
- Author
-
Matthew J. Longley, William C. Copeland, and Eric D. Sullivan
- Subjects
0301 basic medicine ,DNA Replication ,Mitochondrial DNA ,Mitochondrial Diseases ,DNA polymerase ,Ultraviolet Rays ,DNA and Chromosomes ,Biochemistry ,DNA, Mitochondrial ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Polymerase ,030102 biochemistry & molecular biology ,biology ,DNA synthesis ,Chemistry ,DNA replication ,Cell Biology ,Cell biology ,DNA Polymerase gamma ,Mitochondria ,G-Quadruplexes ,030104 developmental biology ,biology.protein ,Biocatalysis ,Replisome ,DNA - Abstract
Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.
- Published
- 2020
38. Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage
- Author
-
Amy Y. Liu, Yu Xu, Bei Liu, Hashim M. Al-Hashimi, Akanksha Manghrani, Honglue Shi, and Uyen Pham
- Subjects
0301 basic medicine ,Purine ,DNA repair ,DNA damage ,Stereochemistry ,Base pair ,genetic processes ,information science ,DNA and Chromosomes ,Sulfuric Acid Esters ,Biochemistry ,Primer extension ,Nucleobase ,03 medical and health sciences ,chemistry.chemical_compound ,Dimethyl sulfate ,Transcription (biology) ,heterocyclic compounds ,Molecular Biology ,Base Pairing ,030102 biochemistry & molecular biology ,Cell Biology ,DNA ,Methylation ,DNA Methylation ,030104 developmental biology ,chemistry ,biological sciences ,health occupations ,Biophysics - Abstract
As the Watson-Crick faces of nucleobases are protected in double-stranded DNA (dsDNA), it is commonly assumed that deleterious alkylation damage to the Watson-Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson-Crick faces of nucleobases has been reported in dsDNAin vitrothrough mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to single-stranded DNA (ssDNA) has not been quantified. Watson-Crick base-pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base-pairs that expose the Watson-Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson-Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson-Crick A-T base-pairs in dsDNA only conferred ~130-fold protection against adenine-N1 methylation and this protection was reduced to ~40-fold for A(syn)-T Hoogsteen base-pairs embedded in a DNA-drug complex. Our results indicate that Watson-Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base-pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairsin vitroand also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-widein vivo.
- Published
- 2020
39. Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization
- Author
-
Steven E. Bates, Ahmad Besaratinia, Stella Tommasi, and Andrew W Caliri
- Subjects
0301 basic medicine ,senescence ,mouse embryonic fibroblasts ,DNA mismatch repair ,Mutant ,immortalization ,medicine.disease_cause ,Medical and Health Sciences ,Biochemistry ,Transgenic ,Mice ,2.1 Biological and endogenous factors ,oxidative stress ,Aetiology ,Transversion ,Cells, Cultured ,Cellular Senescence ,reactive oxygen species ,Mutation ,Cultured ,Transition (genetics) ,Chemistry ,Biological Sciences ,photodynamic therapy ,8-Hydroxy-2'-Deoxyguanosine ,transversion ,mutagenesis ,Senescence ,Biochemistry & Molecular Biology ,Cells ,Mice, Transgenic ,DNA and Chromosomes ,03 medical and health sciences ,oncogenesis ,Genetics ,medicine ,cancer ,Animals ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,Mutagenesis ,Cell Biology ,Molecular biology ,030104 developmental biology ,Chemical Sciences ,DNA damage ,8-oxoguanine ,Carcinogenesis ,Immortalised cell line - Abstract
To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.
- Published
- 2020
40. DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain
- Author
-
Erika Miyazaki, Chihiro Hayashi, Shogo Ozaki, Tsutomu Katayama, and Yoshito Abe
- Subjects
0301 basic medicine ,Models, Molecular ,genetic processes ,Replication Origin ,DNA and Chromosomes ,Biochemistry ,Protein–protein interaction ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Amino Acid Sequence ,Molecular Biology ,dnaB helicase ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Escherichia coli Proteins ,DNA replication ,Helicase ,Cell Biology ,DnaA ,030104 developmental biology ,chemistry ,Replication Initiation ,biology.protein ,Biophysics ,health occupations ,bacteria ,dnaC ,DnaB Helicases ,DNA - Abstract
The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB–DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB–DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe(46) in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I–binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo. On the basis of these findings, we propose that DnaB Leu(160) interacts with DnaA domain I Phe(46). DnaB Leu(160) is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I–bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA–DnaB–DnaC complex, which could be relevant to the process of DnaB loading onto oriC.
- Published
- 2020
41. The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair
- Author
-
Cody M. Rogers, Nicholas J. Buehler, Sabine Wenzel, Matthew L. Bochman, Yuichiro Takagi, Francisco Martínez-Márquez, Samuel Parkins, Sua Myong, and Chun-Ying Lee
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,DNA Repair ,DNA repair ,DNA damage ,Saccharomyces cerevisiae ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein–DNA interaction ,Molecular Biology ,Polymerase ,Nuclease ,Endodeoxyribonucleases ,030102 biochemistry & molecular biology ,biology ,RecQ Helicases ,Chemistry ,DNA replication ,Helicase ,Cell Biology ,DNA ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,biology.protein ,DNA Damage - Abstract
DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.
- Published
- 2020
42. A non-canonical role for the DNA glycosylase NEIL3 in suppressing APE1 endonuclease-mediated ssDNA damage
- Author
-
Anh Ha, Yunfeng Lin, and Shan Yan
- Subjects
0301 basic medicine ,Genome instability ,DNA damage ,DNA repair ,Amino Acid Motifs ,Xenopus Proteins ,DNA and Chromosomes ,Biochemistry ,AP endonuclease ,03 medical and health sciences ,chemistry.chemical_compound ,Endonuclease ,Xenopus laevis ,DNA-(Apurinic or Apyrimidinic Site) Lyase ,Animals ,DNA Breaks, Single-Stranded ,Molecular Biology ,N-Glycosyl Hydrolases ,Ovum ,030102 biochemistry & molecular biology ,biology ,Cell Biology ,Base excision repair ,Cell biology ,Oxidative Stress ,030104 developmental biology ,chemistry ,DNA glycosylase ,biology.protein ,DNA - Abstract
The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein–protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.
- Published
- 2020
43. Impact of 1,N(6)-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair
- Author
-
F. Peter Guengerich and Pratibha P. Ghodke
- Subjects
0301 basic medicine ,Genome instability ,DNA Replication ,Ribonucleotide ,Adenosine ,DNA Repair ,DNA polymerase ,DNA damage ,Ribonuclease H ,DNA-Directed DNA Polymerase ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,DNA adduct ,Humans ,Molecular Biology ,Polymerase ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Cell Biology ,Reverse transcriptase ,Cell biology ,030104 developmental biology ,biology.protein ,Guanosine Triphosphate ,DNA ,DNA Damage - Abstract
Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N(6)-ethenoadenosine (1,N(6)-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N(6)-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N(6)-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N(6)-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N(6)-ϵrA. We also found that RNase H2 recognizes 1,N(6)-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N(6)-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N(6)-ϵrA is incompletely incised by RNase H2.
- Published
- 2020
44. Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB
- Author
-
Michael R. Baldwin, Patrick J. O’Brien, and Suzanne J. Admiraal
- Subjects
0301 basic medicine ,DNA, Bacterial ,DNA Repair ,DNA damage ,DNA repair ,AlkB ,DNA, Single-Stranded ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,DNA adduct ,Escherichia coli ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase ,Escherichia coli Proteins ,AlkB Enzymes ,Cell Biology ,Adaptive response ,DNA ,030104 developmental biology ,DNA glycosylase ,biology.protein ,Nucleic acid ,AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase - Abstract
AlkB is a bacterial Fe(II)– and 2-oxoglutarate–dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N(6)-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.
- Published
- 2020
45. Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability
- Author
-
Michael A. Freitas, Prabakaran Nagarajan, Callie M. Lovejoy, Paula A. Agudelo Garcia, Dongju Park, Mark R. Parthun, and Liudmila V. Popova
- Subjects
0301 basic medicine ,DNA Replication ,DNA and Chromosomes ,Biochemistry ,Cell Line ,Histone H4 ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,Animals ,Nucleosome ,Molecular Biology ,Histone Acetyltransferases ,MRE11 Homologue Protein ,030102 biochemistry & molecular biology ,biology ,Chemistry ,DNA replication ,Cell Biology ,DNA ,Histone acetyltransferase ,Chromatin Assembly and Disassembly ,DNA Replication Fork ,Chromatin ,Cell biology ,030104 developmental biology ,Histone ,biology.protein ,Replisome ,HAT1 - Abstract
The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
- Published
- 2020
46. Replication protein A binds RNA and promotes R-loop formation
- Author
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Andrey G. Baranovskiy, Tahir H. Tahirov, Farid A. Kadyrov, Lyudmila Y. Kadyrova, Alexander V. Mazin, Srinivas Somarowthu, and Olga M. Mazina
- Subjects
DNA Replication ,0301 basic medicine ,DNA repair ,DNA polymerase ,R-loop ,DNA and Chromosomes ,Biochemistry ,complex mixtures ,chemistry.chemical_compound ,03 medical and health sciences ,0302 clinical medicine ,Replication Protein A ,Humans ,Nucleic acid structure ,Molecular Biology ,Replication protein A ,Polymerase ,030304 developmental biology ,0303 health sciences ,030102 biochemistry & molecular biology ,biology ,Chemistry ,DNA replication ,RNA ,DNA ,Cell Biology ,Cell biology ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,biology.protein ,Human genome ,R-Loop Structures ,Homologous recombination ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Replication protein A (RPA), a major eukaryotic ssDNA-binding protein, is essential for all metabolic processes that involve ssDNA, including DNA replication, repair, and damage signaling. To perform its functions, RPA binds ssDNA tightly. In contrast, it was presumed that RPA binds RNA weakly. However, recent data suggest that RPA may play a role in RNA metabolism. RPA stimulates RNA-templated DNA repair in vitro and associates in vivo with R-loops, the three-stranded structures consisting of an RNA-DNA hybrid and the displaced ssDNA strand. R-loops are common in the genomes of pro- and eukaryotes, including humans, and may play an important role in transcription-coupled homologous recombination and DNA replication restart. However, the mechanism of R-loop formation remains unknown. Here, we investigated the RNA-binding properties of human RPA and its possible role in R-loop formation. Using gel-retardation and RNA/DNA competition assays, we found that RPA binds RNA with an unexpectedly high affinity (K(D) ≈ 100 pm). Furthermore, RPA, by forming a complex with RNA, can promote R-loop formation with homologous dsDNA. In reconstitution experiments, we showed that human DNA polymerases can utilize RPA-generated R-loops for initiation of DNA synthesis, mimicking the process of replication restart in vivo. These results demonstrate that RPA binds RNA with high affinity, supporting the role of this protein in RNA metabolism and suggesting a mechanism of genome maintenance that depends on RPA-mediated DNA replication restart.
- Published
- 2020
47. PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection
- Author
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Vanesa Lafarga, Carmen Morales, Diego Megías, Miriam Rodríguez-Corsino, Jan-Michael Peters, Juan Méndez, Sara Rodriguez-Acebes, Miguel Ruiz-Torres, David A. Cisneros, Ana Losada, Ministerio de Economía, Industria y Competitividad (MINECO), European Commision, Ministerio de Economía, Industria y Competitividad (España), and Unión Europea. Comisión Europea
- Subjects
0301 basic medicine ,Genome instability ,DNA Replication ,fork stalling ,DNA repair ,Chromosomal Proteins, Non-Histone ,replication stress ,Cellbiologi ,fork protection ,cohesin ,Cell Cycle Proteins ,Biology ,DNA and Chromosomes ,DNA replication ,Biochemistry ,fork reversal ,Replication fork protection ,03 medical and health sciences ,Mice ,Animals ,Humans ,Molecular Biology ,Cells, Cultured ,BRCA2 Protein ,MRE11 Homologue Protein ,030102 biochemistry & molecular biology ,Cohesin ,replisome ,Biochemistry and Molecular Biology ,Nuclear Proteins ,Cell Biology ,genomic instability ,BRCA2 ,Chromatin ,Cell biology ,Establishment of sister chromatid cohesion ,DNA-Binding Proteins ,030104 developmental biology ,microscopy ,Replisome ,ATPases Associated with Diverse Cellular Activities ,cell cycle ,Rad51 Recombinase ,biological phenomena, cell phenomena, and immunity ,Biokemi och molekylärbiologi ,HeLa Cells ,Transcription Factors - Abstract
Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER Grants BFU2013-48481-R and BFU2016-79841-R (to A. L.) and BFU2016-80402-R (to J. M.) and by FPI "Severo Ochoa" fellowships (to C. M. and M. R.-T.). This work was also supported by funding from Boehringer Ingelheim Fonds (to M. R.-T.). The authors declare that they have no conflicts of interest with the contents of this article. Sí
- Published
- 2020
48. Genetic Evidence for the Involvement of Mismatch Repair Proteins, PMS2 and MLH3, in a Late Step of Homologous Recombination
- Author
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Islam Shamima Keka, Shunichi Takeda, Masataka Tsuda, Raphael Guerois, Maminur Rahman, Hiroyuki Sasanuma, Mohiuddin Mohiuddin, Jessica Andreani, Kousei Yamada, Valérie Borde, Jean-Baptiste Charbonnier, Kyoto University, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), Kyoto University [Kyoto], and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU)
- Subjects
G2 Phase ,0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA Repair ,Resolvase ,[SDV]Life Sciences [q-bio] ,RAD51 ,homologous recombination ,MLH3 ,DNA and Chromosomes ,Biochemistry ,Piperazines ,endonuclease ,Cell Line ,03 medical and health sciences ,Endonuclease ,PMS2 ,Holliday junction ,Humans ,DNA Breaks, Double-Stranded ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,mutL homolog 3 (MLH3) ,Molecular Biology ,mutL homolog 1 (MLH1) ,Mismatch Repair Endonuclease PMS2 ,DNA, Cruciform ,030102 biochemistry & molecular biology ,biology ,Chemistry ,MUS81 ,Cell Biology ,Joint Molecules ,Molecular biology ,digestive system diseases ,MutL Proteins ,030104 developmental biology ,Gamma Rays ,Mutation ,biology.protein ,Phthalazines ,Camptothecin ,DNA mismatch repair ,GEN1 ,Homologous recombination - Abstract
International audience; Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in non-meiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3-/- and PMS2-/- mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3-/- and MLH3DN/DN cells showed a very similar phenotype, a 2.5 times decrease in the frequency of heteroallelic HR-dependent repair of a restriction-enzyme-induced double-strand breaks. PMS2-/- and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of g-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and three times decrease in the number of cisplatin-induced sister chromatid exchange (SCE). The ectopic expression of the Gen1 HJ resolvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.
- Published
- 2020
49. Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication
- Author
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Kazuo Shin-ya, Michael Leffak, Helmut Hanenberg, Joanna Barthelemy, Eric J. Romer, French J. Damewood, Rujuta Yashodhan Gadgil, Caitlin C. Goodman, and S. Dean Rider
- Subjects
0301 basic medicine ,Genome instability ,DNA Replication ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA repair ,DNA damage ,Medizin ,Biology ,DNA and Chromosomes ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Tumor Cells, Cultured ,Humans ,DNA Breaks, Double-Stranded ,Molecular Biology ,030102 biochemistry & molecular biology ,DNA replication ,Microsatellite instability ,Cell Biology ,DNA ,medicine.disease ,Endonucleases ,Molecular biology ,nervous system diseases ,DNA-Binding Proteins ,030104 developmental biology ,chemistry ,Microsatellite ,Human genome ,HeLa Cells ,Microsatellite Repeats - Abstract
Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG)(n) and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)(100) microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)(100) and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)(100) lagging-strand template. (CTG/CAG)(n) chromosome fragility is repeat length–dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)(100) microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)(100) microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure–prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.
- Published
- 2020
50. The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair
- Author
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Christopher D. Putnam, Richard D. Kolodner, and William J. Graham
- Subjects
0301 basic medicine ,DNA mismatch repair ,MutS ,DNA endonuclease ,S. cerevisiae ,Crystallography, X-Ray ,Medical and Health Sciences ,Biochemistry ,chemistry.chemical_compound ,Endonuclease ,0302 clinical medicine ,Adenosine Triphosphate ,Crystallography ,DNA clamp ,biology ,Biological Sciences ,exonuclease 1 ,Cell biology ,DNA-Binding Proteins ,MutS Homolog 2 Protein ,Mlh1-Pms1 ,MutL Protein Homolog 1 ,mutagenesis ,Protein Binding ,Biochemistry & Molecular Biology ,congenital, hereditary, and neonatal diseases and abnormalities ,Saccharomyces cerevisiae Proteins ,DNA repair ,1.1 Normal biological development and functioning ,Saccharomyces cerevisiae ,DNA and Chromosomes ,DNA replication ,03 medical and health sciences ,Underpinning research ,cerevisiae ,Molecular Biology ,neoplasms ,Msh2-Msh6 ,Mutagenesis ,nutritional and metabolic diseases ,Cell Biology ,digestive system diseases ,DNA binding protein ,030104 developmental biology ,chemistry ,MSH2 ,Chemical Sciences ,X-Ray ,biology.protein ,Generic health relevance ,030217 neurology & neurosurgery ,DNA - Abstract
DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.
- Published
- 2018
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