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TOX (thymocyte selection-associated HMG BOX) is a member of a family of transcriptional factors that contain the highly conserved high mobility group box (HMG-box) region. Increasing studies have shown that TOX is involved in maintaining tumors and promoting T cell exhaustion. In this review, we summarized the biological functions of TOX and its contribution as related to lymphocytic malignancies. We also discussed the potential role of TOX as an immune biomarker and target in immunotherapy for hematological malignancies.

The sex-determining region Y (SRY) gene is a key gene involved in male sex differentiation and development. Patients with 46,XY disorders of sex development related to mutations in the high mobility group (HMG) box typically present with complete gonadal dysgenesis. In this study, we report a case of novel missense mutation c.T281G within the HMG domain of SRY in a 15-year-old patient of the female gender with 46,XY partial gonadal dysgenesis (PGD). The novel missense mutation caused the substitution of codon 94 for leucine in the HMG box of the SRY protein with an arginine codon. Leucine and arginine are aliphatic amino acids, and three-dimensional protein structure prediction revealed only slight structural changes in the SRY protein. Thus, the SRY protein had maintained some of its functions, and the patient presented with PGD. In conclusion, we identified a novel SRY mutation in a patient with 46,XY PGD. Based on the protein model, we believe that the mutation in the HMG domain helped to maintain the partial function of the SRY protein. The condition of our patient differed from the well-known 46,XY complete gonadal dysgenesis caused by mutations in the HMG region. In fact, this is the first case of 46,XY PGD caused by mutations in the HMG region to be reported, and therefore, our experience has expanded the mutation spectrum of the SRY gene. Furthermore, the present case demonstrates that mutations located in the HMG domain of SRY gene cannot be ruled out in patients with a clinical diagnosis of 46,XY PGD.

Hydroxyurea (HU) activates the γ-globin gene, resulting in increased Hb F synthesis. The SOX6 gene is a member of the Sox (Sry-type HMG box) family of transcription factors, characterized by minor ...

High mobility group box (HMGB) members are DNA binding proteins with varied functions present across kingdoms. The mechanism by which HMGBs with varying number of HMG boxes are able to carry out similar functions, are poorly understood. Moreover, how non-canonical DNAs are recognized by HMGB proteins is not clear. To address these, we carried out detailed biochemical and computational studies to characterize two HMGB members- Kinetoplast associated protein (KAP6) of Trypanosoma and High mobility group protein 1 (HMO1) from yeast. Here, we report that KAP6 binds non-canonical DNAs tighter than B-form DNA. Among non-canonical DNAs, KAP6 has the highest affinity for splayed and flap structures, but least for Holliday Junction (HJ). In contrast, HMO1 binds tighter to HJ. Computational analysis show that the secondary structural elements involved in DNA interaction are conserved in HMGB members KAP6 and mitochondrial transcription factor A. Simulation analyses revealed that the ~90° bend in DNA induced by KAP6 HMG box is a result of two ~45° bends, by Helix 1 and Helix 2 of the protein. Our data also suggests that the orthologs of HMO1 and KAP6 are oligomers in solution, which could be necessary for their functioning such as DNA bending and looping.

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

SRY-related HMG box (SOX)21, one of the most highly expressed transcription inhibitors in the central nervous system (CNS), is involved in neurogenesis-related transcription and proliferation, which are associated with certain neurological disorders. However, it is the role of SOX21 in the pathogenesis of epilepsy remains unclear. In this study, our aim was to examine the expression and function of SOX21 in patients with temporal lobe epilepsy (TLE), as well as pentylenetetrazol (PTZ)-kindled rats, and to identify the possible roles of SOX21 in epileptogenesis. We found that SOX21 localized in neurons is upregulated, especially in TLE patients. SOX21 is present in the hippocampus or adjacent temporal cortex in the PTZ-kindled epileptic rat model. In addition, the P-gpMAbNano-structured material (PNM) nanoparticles carrying anti-epileptic drugs (AEDs) were injected into the epileptic model rats using an intravenous injection. The expression of tumor necrosis factor peptide in the rats was detected to verify whether the drug-carrying nanoparticles could bypass macrophages and reach the target for treatment. We also found an interaction between SOX21 and SOX2 in PTZ-kindled rats. These results indicate that SOX21 is mainly located in neurons and may regulate the pathogenesis of epilepsy, possibly in association with SOX2. Moreover, PNM nanoparticles equipped with AEDs can reach the target through macrophages in vivo, providing a new approach for the clinical treatment of epilepsy.

T-cell factor 4 (TCF-4) is determined to play a crucial role in Wnt/β-catenin signaling pathway activation. The mutations and alternative splice isoforms of TCF-4 can cause cancers and other diseases. The high-mobility group (HMG) box domain of TCF-4 contributes to interacting with DNA motif for transcriptional regulation. However, the impact of the mutations within HMG box of TCF-4 on the genomic binding pattern is poorly investigated. Herein, we generated non–small cell lung cancer (NSCLC) cell line A549 with stably overexpressed TCF-4 with HMG box hot spot mutation (10th exon partial deletion), and conducted TCF-4 and β-catenin chromatin immunoprecipitation sequence to explore the differential genomic binding patterns. Our results revealed that TCF-4 lost 19365 but gained 1724 peaks, and β-catenin lost 4035 but gained 5287 peaks upon mutant TCF-4 compared with the wild type (log2FC > 1 or

In contrast to oligodeoxynucleotides, protein:DNA complexes crystallize from a broad range of precipitants and conditions, much as proteins by themselves. There are, however, a number of factors that should be considered, at least in the early stages of cocrystallization attempts. These include the length and construction of the oligodeoxynucleotide itself, a pH near or below neutrality, a stoichiometric excess of DNA, and di- and polyvalent cations. By far the more important of these factors are the length of the DNA fragment and the nature of the terminal nucleotides. Unfortunately, experience suggests that, in general, the length and construction of the DNA fragment for optimal crystal growth cannot be predicted in advance. A systematic search of cocrystallization conditions with different DNA fragments will normally be required. It is important to avoid sequences that provide possible subsites within the fragment or at junctions between fragments related by translations. Sample purity, especially that of the DNA, can also have important effects. Detailed protocols for the purification of chemically synthesized fragments are suggested. Finally, a set of conditions for initial cocrystallization trials with a new DNA binding protein is suggested.

The present study aimed to search for chicken abdominal fat deposition-related polymorphisms within RB1 and to provide functional evidence for significantly associated genetic variants. Association analyses showed that 11 single nucleotide polymorphisms (SNPs) in intron 17 of RB1, were significantly associated with both abdominal fat weight (P G repressed the transcriptional efficiency of RB1 in vitro, through binding nuclear factor-kappa B (NF-KB) and SRY-related HMG box protein 2 (SOX2). Furthermore, RB1 mRNA expression levels in the abdominal fat tissue of individuals with the A/A genotype of g.32828A>G were lower than those of individuals with the G/G genotype. Collectively, we propose that the intronic SNP g.32828A>G of RB1 is an obesity-associated variant that directly affects binding with NF-KB and SOX2, leading to changes in RB1 expression which in turn may influence chicken abdominal fat deposition.

Capicua (CIC) is a transcriptional repressor and functions downstream of the receptor tyrosine kinase (RTK) signaling pathway. Somatic mutations found in the HMG-box DNA binding domain in CIC have been implicated in several cancers such as oligodendroglioma, oligoastrocytoma, and adenocarcinoma. However, the molecular basis of the DNA binding of CIC and the effect of the somatic mutations found in cancers on DNA binding have not been investigated. Here, we report the crystal structure of the HMG-box domain of CIC complexed with its target DNA, the promoter of Ets Translocation Variant 5 (ETV5). The structure shows that the HMG-box domain has an L-shaped structure and recognizes the minor groove leading to DNA bending. Our structure combined with an electrophoretic mobility shift assay (EMSA) revealed that cancer-associated mutations in the HMG-box domain abrogate the interaction with DNA. These results provide the molecular insight into the DNA binding of CIC and reveal the effects of carcinogenic mutations on DNA binding.

SOX4, SOX11 and SOX12 are the members of group C of Sry-related HMG box proteins. Inspite of being discovered more than 15 years ago, their molecular properties and functions are not completely understood. Humans and mice contain 20 SOX genes and 8 are present in Drosophila. Our phylogenetic tree result shows that SOX4 and SOX12 in Primates and Rodentia are more closely related to each other with a few exceptions. Phylogenetic tree based on the amino acid sequences of HMG box domain showed that SOX4 in Homo sapiens belongs to the same cluster and is closely related with SOX11 of Homo sapiens and Mus musculus. Protein compositional result reveals that random coil and alpha helix are the most abundant forms in all the cases. A negative GRAVY value indicates the hydrophilic nature of the protein, with its sub-cellular localization predicted to be within the nucleus, hence they cannot be a membrane protein. Analysis of SOXC molecular features shows that SOX4, SOX11 and SOX12 are single-exon genes, with N-terminal Sry-related HMG box (SOX) domain and a C-terminus transactivation domain (TAD). Their expression analysis revealed some overlapping expression pattern in some cancers like breast adenocarcinoma, esophageal adenocarcinoma, squamous cell lung carcinoma etc. Studies looking at the sites where SOX genes are located may provide useful information on their potential role in cancers. In humans, SOX4, SOX11 and SOX12 are located on chromosome 6, 2 and 20 respectively and in mouse they are located on chromosome 13, 12 and 2 respectively.

The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

High mobility group proteins (HMGs) are the most abundant nuclear proteins next to histones and are robustly expressed across tissues and organs. HMGs can uniquely bend or bind distorted DNA, and are central to such processes as transcription, recombination, and DNA repair. However, their dynamic association with chromatin renders capturing HMGs on chromosomes challenging. Recent work has changed this and now implicates these factors in spatial genome organization. Here, I revisit older and review recent literature to describe how HMGs rewire spatial chromatin interactions to sustain homeostasis or promote cellular aging. I propose a 'rheostat' model to explain how HMG-box proteins (HMGBs), and to some extent HMG A proteins (HMGAs), may control cellular aging and, likely, cancer progression.

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a humanSRYtransgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each ofSRY’s three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to humanSRYand the causes of DSD, and present a versatile and powerful system in which to demonstrate causality ofSRYvariations in DSD, to functionally study theSRYvariation database, and to characterize new pathogenicSRYvariations found in DSD.

OBJECTIVES: Glycemic dysregulation is one of the major metabolic disorders, which has long been a significant public health issue. The liver plays a pivotal role in the maintenance of blood glucose homeostasis. Previous studies indicate that upon refeeding a high carbohydrate diet, the expression of transcription factor HMG box-containing protein 1 (HBP1) was elevated in mice livers, suggesting a role of HBP1 in carbohydrate metabolism. Therefore, the objective of the current study was to understand the molecular mechanisms through which HBP1 regulates glucose generation in the liver. METHODS: Both in vivo HBP1 knockdown mice and in vitro HepG2 cell line were employed as the experimental models. RESULTS: First, we observed that overnight fasting led to increased PEPCK but decreased HBP1 expression in mice livers, and subsequent addition of insulin reversed the expression pattern. More importantly, HBP1 knockout (KO) mice displayed a significantly higher blood glucose level (173 mg/dL) than that of the controls (98 mg/dL). Also, HBP1 KO led to impaired OGTT (oral glucose tolerance test) and ITT (insulin tolerance test). These data suggest that HBP1 might have a role in the regulation of gluconeogenesis in the liver. To unveil the molecular mechanism by which HBP1 regulates glucose homeostasis, we examined its role in gluconeogenesis. Administration of gluconeogenic stimulators glucagon (100 nM) and cAMP (0.5 ng/mL) resulted in increased expression of Phosphoenolpyruvate carboxykinase (PEPCK; encoded by the PCK1 gene), a key enzyme in gluconeogenesis, but decreased HBP1 expression in HepG2 cells. Last, HBP1 siRNA-mediated mRNA disruption led to elevated PEPCK expression, whereas ectopic expression of HBP1 (pcDNA3-HBP1-Flag) significantly suppressed it. CONCLUSIONS: In conclusion, our data indicate that HBP1 might negatively regulate glucose production and support HBP1 as a novel biological regulator of blood glucose homeostasis. FUNDING SOURCES: This work was supported by the grant to MOST 108–2320-B-039–051-MY3 C-Y Huang.

High Mobility Group Box 1 protein (HMGB1) is an abundant protein with multiple functions in cells, acting as a DNA chaperone and damage-associated molecular pattern molecule. It represents an attractive target for the treatment of inflammatory diseases and cancers. The plant natural product glycyrrhizin (GLR) is a well-characterized ligand of HMGB1 and a drug used to treat diverse liver and skin diseases. The drug is known to bind to each of the two adjacent HMG boxes of the non-glycosylated protein. In cells, HMGB1 is N-glycosylated at three asparagine residues located in boxes A and B, and these N-glycans are essential for the nucleocytoplasmic transport of the protein. But the impact of the N-glycans on drug binding is unknown. Here we have investigated the effect of the N-glycosylation of HMGB1 on its interaction with GLR using molecular modelling, after incorporation of three N-glycans on a Human HMGB1 structure (PDB code 2YRQ). Sialylated bi-antennary N-glycans were introduced on the protein and exposed in a folded or an extended conformation for the drug binding study. The docking of the drug was performed using both 18α- and 18β-epimers of GLR and the conformations and potential energy of interaction (ΔE) of the different drug-protein complexes were compared. The N-glycans do not shield the drug binding sites on boxes A and B but can modulate the drug-protein interaction, via both direct and indirect effects. The calculations indicate that binding of 18α/β-GLR to the HMG box is generally reduced when the protein is N-glycosylated vs. the non-glycosylated protein. In particular, the N-glycans in an extended configuration significantly weaken the binding of GLR to box-B. The effects of the N-glycans are mostly indirect, but in one case a direct contact with the drug, via a carbohydrate-carbohydrate interaction, was observed with 18β-GLR bound to Box-B of glycosylated HMGB1. For the first time, it is shown (at least in silico) that N-glycosylation, one of the many post-translational modifications of HMGB1, can affect drug binding.

The goal of this study was to clone and characterize complete cDNA sequences of three important development-relevant genes of yellow catfish Pelteobagrus fulvidraco, including lrp6, sox9a1 and fgfr2c, and explore their transcriptional responses in several tissues of P. fulvidraco to high fat diet. The predicted amino acid sequences of P. fulvidraco Lrp6, Sox9a1 and Fgfr2c contained all of the conserved structural features that were characteristic of these genes in other species, including YWTD domains, EGF-like repeats, LDLR ligand binding repeats, PPSP repeats motifs, HMG box, TA-binding functional domain, Ig I-III and PTK I-II. The mRNAs of the three genes were expressed in various tissues, but their mRNA levels varied among tissues. Compared to the control, high fat diet tended to down-regulate the mRNA expression of sox9a1 and fgfr2c in mesenteric fat, liver and ovary, and up-regulate their mRNA levels in muscle and kidney; in contrast, high fat diet down-regulated lrp6 mRNA levels in the ovary and muscle, but had no significant effects on lrp6 mRNA expression in mesenteric fat, liver and kidney. Our findings provide the first data about their expression responses to dietary lipid in teleosts and reinforce the multiple functions at the molecular level.

Glioblastoma multiforme (GBM) is the most malignant neuroepithelial primary brain tumor and its mean survival time is 15 months after diagnosis. This study undertook to investigate the genome-wide and transcriptome-wide analyses of human high mobility group box (HMG-box) TF (transcript factor) families / HOX, TOX, FOX, HMG and SOX gene families, and their relationships to GBM. According to the TCGA-GBM profile analysis, differentially expressed HOX, FOX, HMG and SOX gene families (62 DEmRNA) were found in this study. We also analyzed DEmRNA (HMG-box related genes) co-expressed eight DElncRNA in GBM, and constructed a ceRNA network analysis as well. We constructed 50 DElncRNA-DEmiRNA-DEmRNA (HMG-box related genes) pairs between GBM and normal tissues. Then, risk genes SOX6 and SOX21 expression were correlated with immune infiltration levels in GBM. SOX6 also had a strong association with MAPT, GSK3B, FYN and DPYSL4, suggesting that they might be functional members in GBM.

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1–2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1–2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1 ∆ HMG completely lacking the HMG-box domain was studied. SSRP1 ∆ HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1 ∆ HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1–1 mutant. SSRP1 ∆ HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.

High-mobility group (HMG)-box proteins are involved in chromatin organization in eukaryotes, especially in sex determination and regulation of mitochondrial DNA compaction. Although a novel HMG-box protein, PoxHmbB, had been initially identified to be required for filter paper cellulase activity by Penicillium oxalicum, the biological roles of HMG-box proteins in biomass-degrading enzyme production have not been systematically explored. The P. oxalicum mutant ∆PoxHmbB lost 34.7-86.5% of cellulase (endoglucanase, p-nitrophenyl-β-cellobiosidase, and p-nitrophenyl-β-glucopyranosidase) activities and 60.3% of xylanase activity following Avicel induction, whereas it exhibited about onefold increase in amylase activity following soluble corn starch induction. Furthermore, ∆PoxHmbB presented delayed conidiation and hyphae growth. Transcriptomic profiling and real-time quantitative reverse transcription-PCR revealed that PoxHmbB regulated the expression of major genes encoding plant biomass-degrading enzymes such as PoxCel7A-2, PoxCel5B, PoxBgl3A, PoxXyn11B, and PoxGA15A, as well as those involved in conidiation such as PoxBrlA. In vitro binding experiments further confirmed that PoxHmbB directly binds to the promoter regions of these major genes. These results further indicate the diversity of the biological functions of HMG-box proteins and provide a novel and promising engineering target for improving plant biomass-degrading enzyme production in filamentous fungi.

The homeodomain-containing transcription factor Anf (also known as Rpx/Hesx1 in mammals) plays an important role during the forebrain development in vertebrates. Here we demonstrate the ability of the Xenopus laevis Anf, Xanf1/Hesx1, to directly bind SRY-related HMG-box-containing transcription factor SoxD/Sox15 and to cooperate with the latter during regulating of the expression of Xanf1/Hesx1 own gene. As we have shown by GST pull-down, EMSA and the luciferase reporter assays, Xanf1/Hesx1 and SoxD/Sox15 bind the Xanf1/Hesx1 promoter region counteracting the inhibitory effect of Xanf1/Hesx1 alone. This finding explains how Xanf1/Hesx1 could escape the repressive activity of its own protein during early patterning of the forebrain rudiment.

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.

Accurate repair of DNA breaks is essential to maintain genome integrity and cellular fitness. Sgs1, the sole member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is important for both early and late stages of homology-dependent repair. Its large number of physical and genetic interactions with DNA recombination, repair, and replication factors has established Sgs1 as a key player in the maintenance of genome integrity. To determine the significance of Sgs1 binding to the strand-exchange factor Rad51, we have identified a single amino acid change at the C-terminal of the helicase core of Sgs1 that disrupts Rad51 binding. In contrast to an SGS1 deletion or a helicase-defective sgs1 allele, this new separation-of-function allele, sgs1-FD, does not cause DNA damage hypersensitivity or genome instability, but exhibits negative and positive genetic interactions with sae2Δ, mre11Δ, exo1Δ, srs2Δ, rrm3Δ, and pol32Δ that are distinct from those of known sgs1 mutants. Our findings suggest that the Sgs1-Rad51 interaction stimulates homologous recombination (HR). However, unlike sgs1 mutations, which impair the resection of DNA double-strand ends, negative genetic interactions of the sgs1-FD allele are not suppressed by YKU70 deletion. We propose that the Sgs1-Rad51 interaction stimulates HR by facilitating the formation of the presynaptic Rad51 filament, possibly by Sgs1 competing with single-stranded DNA for replication protein A binding during resection.

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function.

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. In contrast to the nucleosome core histone proteins, the role of H1 in compacting DNA is not clearly understood. In this study ITC was used to measure the binding constant, enthalpy change, and binding site size for the interactions of H1 0 , or its C-terminal (H1 0 -C) and globular (H1 0 -G) domains to highly polymerized calf-thymus DNA at temperatures from 288 K to 308 K. Heat capacity changes, ΔC p , for these same H1 0 binding interactions were estimated from the temperature dependence of the enthalpy changes. The enthalpy changes for binding H1 0 , H1 0 -C, or H1 0 -G to CT-DNA are all endothermic at 298 K, becoming more exothermic as the temperature is increased. The ΔH for binding H1 0 -G to CT-DNA is exothermic at temperatures above approximately 300 K. Osmotic stress experiments indicate that the binding of H1 0 is accompanied by the release of approximately 35 water molecules. We estimate from our naked DNA titration results that the binding of the H1 0 to the nucleosome places the H1 0 protein in close contact with approximately 41 DNA bp. The breakdown is that the H1 0 carboxyl terminus interacts with 28 bp of linker DNA on one side of the nucleosome, the H1 0 globular domain binds directly to 7 bp of core DNA, and shields another 6 linker DNA bases, 3 bp on either side of the nucleosome where the linker DNA exits the nucleosome core.

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to mark genes for repression. We measured the dynamics of PRC2 binding on recombinant chromatin and free DNA at the single-molecule level using total internal reflection fluorescence (TIRF) microscopy. PRC2 preferentially binds free DNA with multisecond residence time and midnanomolar affinity. PHF1, a PRC2 accessory protein of the Polycomblike family, extends PRC2 residence time on DNA and chromatin. Crystallographic and functional studies reveal that Polycomblike proteins contain a winged-helix domain that binds DNA in a sequence-nonspecific fashion. DNA binding by this winged-helix domain accounts for the prolonged residence time of PHF1-PRC2 on chromatin and makes it a more efficient H3K27 methyltranferase than PRC2 alone. Together, these studies establish that interactions with DNA provide the predominant binding affinity of PRC2 for chromatin. Moreover, they reveal the molecular basis for how Polycomblike proteins stabilize PRC2 on chromatin and stimulate its activity.

Transcription factors (TFs) are gene expression regulators that bind to DNA in a sequence-specific manner and determine the functional characteristics of the gene. It is worthwhile to study the unique characteristics of such specific TF-binding pattern in DNA. Sox2 recognizes a 6- to 7-base pair consensus DNA sequence; the central four bases of the binding site are highly conserved, whereas the two to three flanking bases are variable. Here, we attempted to analyze the binding affinity and specificity of the Sox2 protein for distinct DNA sequence patterns via steered molecular dynamics, in which a pulling force is employed to dissociate Sox2 from Sox2-DNA during simulation to study the behavior of a complex under nonequilibrium conditions. The simulation results revealed that the first two stacking bases of the binding pattern have an exclusive impact on the binding affinity, with the corresponding mutant complexes showing greater binding and longer dissociation time than the experimental complexes do. In contrast, mutation of the conserved bases tends to reduce the affinity, and mutation of the complete conserved region disrupts the binding. It might pave the way to identify the most likely binding pattern recognized by Sox2 based on the affinity of each configuration. The α2-helix of Sox2 was found to be the key player in the Sox2-DNA association. The characterization of Sox2's binding patterns for the target genes in the genome helps in understanding of its regulatory functions.

5-Formylcytosine (5fC) is an endogenous DNA modification frequently found within regulatory elements of mammalian genes. Although 5fC is an oxidation product of 5-methylcytosine (5mC), the two epigenetic marks show distinct genome-wide distributions and protein affinities, suggesting that they perform different functions in epigenetic signaling. A unique feature of 5fC is the presence of a potentially reactive aldehyde group in its structure. Here, we show that 5fC bases in DNA readily form Schiff base conjugates with Lys side chains of nuclear proteins in vitro and in vivo. These covalent protein-DNA complexes are reversible (t1/2, 1.8 h), suggesting that they contribute to transcriptional regulation and chromatin remodeling. On the other hand, 5fC mediated DNA-protein cross-links, if present at replication forks or actively transcribed regions, may interfere with DNA replication and transcription.

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions.

A cryo-electron microscopy structure of the chromatin remodelling factor Chd1 bound to a nucleosome leads to a model for DNA translocation by its ATPase motor. The chromatin remodelling factor Chd1 can alter nucleosome positioning and facilitates the passage of RNA polymerase II through nucleosomes. Here, Patrick Cramer and colleagues describe the cryo-electron microscopy structure of full-length yeast Chd1 bound to a nucleosome. The structure reveals that Chd1 detaches two turns of DNA from the histone octamer, with the ATPase motor engaged with DNA at site SHL +2 and Chd1 trapped in a poised state. The authors propose a model for how the ATPase promotes directional translocation and how it is activated by binding of the regulatory double chromodomain to nucleosomal DNA. The structure should aid mechanistic understanding of remodellers in the CHD family across all species and of other remodelling factors that resemble Chd1 in architecture. Chromatin-remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair1. The conserved remodelling factor chromodomain-helicase-DNA binding protein 1(Chd1)2 can shift nucleosomes and induce regular nucleosome spacing3,4,5. Chd1 is required for the passage of RNA polymerase IIthrough nucleosomes6 and for cellular pluripotency7. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses ATP, and a regulatory double chromodomain. Here we report the cryo-electron microscopy structure of Chd1 from the yeast Saccharomyces cerevisiae bound to a nucleosome at a resolution of 4.8 A. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) −7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4, as seen in a recent nucleosome–Snf2 ATPase structure8. Comparisons with published results9 reveal that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocation state in the absence8 or presence, respectively, of transition state-mimicking compounds.

DDK, a conserved serine-threonine protein kinase composed of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7, is essential for DNA replication initiation during S phase of the cell cycle through MCM2-7 helicase phosphorylation. The biological significance of DDK is well characterized, but the full mechanism of how DDK associates with substrates remains unclear. Cdc7 is bound to chromatin in the Saccharomyces cerevisiae genome throughout the cell cycle, but there is little empirical evidence as to specific Cdc7 binding locations. Using biochemical and genetic techniques, this study investigated the specific localization of Cdc7 on chromatin. The Calling Cards method, using Ty5 retrotransposons as a marker for DNA–protein binding, suggests Cdc7 kinase is preferentially bound to genomic DNA known to replicate early in S phase, including centromeres and origins of replication. We also discovered Cdc7 binding throughout the genome, which may be necessary to initiate other cellular processes, including meiotic recombination and translesion synthesis. A kinase dead Cdc7 point mutation increases the Ty5 retrotransposon integration efficiency and a 55-amino acid C-terminal truncation of Cdc7, unable to bind Dbf4, reduces Cdc7 binding suggesting a requirement for Dbf4 to stabilize Cdc7 on chromatin during S phase. Chromatin immunoprecipitation demonstrates that Cdc7 binding near specific origins changes during S phase. Our results suggest a model where Cdc7 is loosely bound to chromatin during G1. At the G1/S transition, Cdc7 binding to chromatin is increased and stabilized, preferentially at sites that may become origins, in order to carry out a variety of cellular processes.

The interaction between the single-stranded DNA and the homologous duplex DNA is essential for DNA homologous repair. Here, we report that parallel triplex structure can form spontaneously between a mechanically extended ssDNA and a homologous dsDNA in protein-free condition. The triplex has a contour length close to that of a B-form DNA duplex and remains stable after force is released. The binding energy between the ssDNA and the homologous dsDNA in the triplex is estimated to be comparable to the basepairing energy in a B-form dsDNA. As ssDNA is in a similar extended conformation within recombinase-coated nucleoprotein filaments, we propose that the parallel triplex may form and serve as an intermediate during recombinase-catalyzed homologous joint formation.

Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation–sequencing has been used to identify and map transcription factor–DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin–interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications.

FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry (ITC) assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and less minor groove contacts in the FOXA2 structure compared to the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.

DNA replication in eukaryotic cells is performed by a multiprotein complex called the replisome, which consists of helicases, polymerases, and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and high mobility group (HMG)-box DNA-binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. Although structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (pol α) to the DNA helicase complex (Cdc45-MCM2–7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homotrimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex, mediated by unknown participants other than Go Ichi Ni San. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway.

Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5’-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3’-phosphotyrosyl(274))-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with lower cleavage site affinity compared to the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ~2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution NMR methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB are underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.

Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation.

The maintenance of genome stability depends on the ability of the cell to repair DNA efficiently. Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. While the role of human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) in the repair of double-stranded breaks has been well established, we have recently shown that it is also essential for the base excision repair (BER) pathway following oxidative DNA damage. However, unlike in DSB repair, the formation of stable hSSB1 oligomers under oxidizing conditions is an important prerequisite for its proper function in BER. In this study, we have used solution-state NMR in combination with biophysical and functional experiments to obtain a structural model of hSSB1 self-oligomerization. We reveal that hSSB1 forms a tetramer that is structurally similar to the SSB from Escherichia coli and is stabilized by two cysteines (C81 and C99) as well as a subset of charged and hydrophobic residues. Our structural and functional data also show that hSSB1 oligomerization does not preclude its function in DSB repair, where it can interact with Ints3, a component of the SOSS1 complex, further establishing the versatility that hSSB1 displays in maintaining genome integrity.

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer's foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.

Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku's requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries.

Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc.

Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45° angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 Å away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a “rail shape” and “flap-on” conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a “septum” that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNAase sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system2-4.

Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8-acetyl-aryl- or C8-aryl-dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2-NH2 group is observed. Although the C8 adducts retain base-pairing properties, base pairing is strongly compromised in the case of the N2 adducts. Here we show crystal structures of two DNA lesions, N2 -acetylnaphthyl-dG and C8-fluorenyl-dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif.

Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7–DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (∼43°), nearly as sharp as that produced by wild-type Cren7 (∼48°), in the bound DNA fragment despite the lack of side chain intercalation. In another duplex DNA fragment, L28F inserts a large hydrophobic side chain deep into the DNA step, but introduces a smaller kink (∼39°) than that formed by the wild-type protein (∼50°). Mutation of Leu28 into methionine yields two protein conformers differing in loop β3–β4 orientation, DNA-binding surface and DNA geometry in the protein–DNA structure. Our results indicate that side chain intercalation is not directly responsible for DNA kinking or bending by Cren7, but plays a critical role in the stabilization of the Cren7–DNA complex. In addition, the flexibility of loop β3–β4 in Cren7, as revealed in the crystal structure of L28M–DNA, may serve a role in the modulation of chromosomal organization and function in the cell. * ds, : double-stranded; EDTA, : ethylenediaminetetraacetic acid disodium salt; H-NS, : histone-like nucleoid-structuring protein; HMG1, : high-mobility group protein 1; HU, : heat unstable protein; IHF, : integrated host factor; r.m.s.d., : root-mean-square deviation; ss, : single-stranded; TBP, : TATA Binding Protein.

The A/B domains of nuclear receptors such as thyroid receptor α (TRα) are considered to be conformationally flexible and can potentially adopt multiple structural conformations. We used intrinsic tryptophan fluorescence quenching and circular dichroism spectroscopy to characterize the unfolding of this A/B domain upon DNA binding to the contiguous DNA-binding domain (DBD). We propose that this allosteric change in A/B domain conformation can allow it to make the multiple interactions with distinct molecular factors of the transcriptional preinitiation complex. We further suggest that by influencing the affinity of the DBD for DNA, A/B domain can fine-tune the recognition of promotor DNA by TRα.

The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA containing capsids from the nucleus, but is also required for optimal replication of viral DNA and its packaging into capsids. Here we report that the UL31 protein from HSV-2 can be recruited to sites of DNA damage by sequences found in its N-terminus. The N-terminus of UL31 contains sequences resembling a poly (ADP-ribose) (PAR) binding motif suggesting that PAR interactions might mediate UL31 recruitment to damaged DNA. Whereas PAR polymerase inhibition prevented UL31 recruitment to damaged DNA, inhibition of signaling through the ataxia telangiectasia mutated DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. This study reveals a previously unrecognized function for UL31 and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.