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This study enhances our understanding of mitochondrial transcription factor A (TFAM) and its pivotal roles in maintaining mitochondrial DNA (mtDNA) integrity through a series of investigations. First, we focused on Glutamic acid 187 (E187) in the β-elimination reaction, which is an essential step in mtDNA repair. Using kinetic analyses and comprehensive computer simulations, we demonstrated how E187 accelerates the reaction rates quantitatively, providing critical insights into the molecular mechanisms that facilitate DNA repair within mitochondria.Next, our study has demonstrated the intrinsic 5’dRp lyase activity of TFAM, observed through in vitro experiments. The activity rate is comparable with that of polymerase β, implying TFAM's vital role in the mitochondrial Base Excision Repair (BER) pathway. Such functionality is particularly significant since it indicates TFAM can reduce the accumulation of toxic DNA repair intermediates. These intermediates if unrepaired, could lead to mitochondrial dysfunction, thus highlighting the broader implications of TFAM in maintaining cellular health and preventing disease progression.Finally, we are working on developing an innovative mass spectrometry method to detect TFAM-DNA-protein complexes (DPCs) in cells. This novel method aims to provide structural insights into the ways TFAM interacts with damaged mtDNA and allow us to detect other protein candidates that are binding with AP-mtDNA under physiological condition. The development of this technique is an important step towards understanding the complex dynamics of mtDNA-protein interactions within mitochondria.Taken together, the above projects help with our understanding of TFAM's molecular interactions and regulatory roles and broaden our knowledge of mtDNA stability. By elucidating the mechanisms that protect mtDNA integrity, our research contributes to the broader scientific understanding of mitochondrial function and its impact on cellular health.

DNA-functionalized gold nanoparticles are one of the most versatile bionanomaterials for biomedical and clinical diagnosis. Herein, we discovered that the performance of DNAzyme cleaving the substrate is highly related to its length. This intriguing phenomenon only appears at the interfaces of DNA-functionalized gold nanoparticles. We systematically investigated the causes of this phenomenon. We conjectured that the DNAzyme with extended nucleotides that do not match its substrate strand is vulnerable to non-specific adsorption, electrostatic repulsion, and steric hindrance. Based on our improved understanding of this phenomenon, we have successfully developed a highly sensitive and specific amplifiable biosensor to detect human apurinic/apyrimidinic endonuclease 1.

The development and application of metal nanoclusters (MNCs) in nucleic acid testing in the past 10 years have been summarized. Fluorescence enhancement and red shift can occur when the G-rich sequence gets close to Ag NCs or the complementary DNA strand hybridizes with Ag NCs tail strand, which can be used to identify the nucleic acid. Ag NCs with the abasic site in DNA duplex can distinguish mutant genes such as cancer suppression gene p53. Ag NCs with auxiliary DNA can be used to detect miR-21, miR-16-5p, miR-19b-3p, and miR-141. Cu NCs/Cu NPs can recognize miRNA-155, miR-21, and miR-let-7d without auxiliary DNA. Au NCs can identify H1N1 gene fragments by fluorescence quenching caused by proximity to the G-rich sequence. Besides, Au NCs can recognize miRNA-21 and let-7a. SsDNA MNCs adsorbed on the surface of GO and CNPs oxide can be used to identify HBV and HIV gene fragments. The addition of enzymes or auxiliary amplification technologies is a popular way to improve probe sensitivity. Ag NCs combined with TAIEA has the best performance and can obtain LOD as low as aM for miRNA.

Cigarette smoking is an established risk factor for oral cancer. The health effects of e-cigarettes are still under investigation but may disturb oral cavity homeostasis and cause lung and cardiovascular diseases. Carcinogens and toxicants in tobacco products and e-cigarettes may damage DNA, resulting in the formation of apurinic/apyrimidinic (AP) sites, and initiation of the carcinogenic process. In this study, we optimized a liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method to analyze AP sites in buccal cell DNA of 35 nonsmokers, 30 smokers, and 30 e-cigarette users. AP sites in e-cigarette users (median 3.3 per 10(7) nts) were significantly lower than in smokers (median 5.7 per 10(7) nts) and nonsmokers (median 6.0 per 10(7) nts). AP sites in smokers were not significantly different from nonsmokers (p > 0.05). The e-cigarette vaporizing solvents propylene glycol and glycerin were tested and did not protect against AP site formation in in vitro control and carcinogen exposed rat liver homogenates. However, propylene glycol may inhibit bacteria in oral cells, resulting in reduced inflammation and related effects, and reduced AP site levels in e-cigarette user DNA. This is the first study to examine AP site formation in e-cigarette users and to evaluate AP sites in human oral cell DNA.

Interstrand DNA cross-links are important in biology, medicinal chemistry, and materials science. Methods for the targeted installation of interstrand cross-links in DNA duplexes may be useful in diverse fields including studies of DNA repair, materials science, and structural biology. Here a simple procedure is reported for the preparation of DNA duplexes containing site-specific, chemically-defined interstrand cross-links. The approach involves sequential reductive amination reactions between diamine linkers and two abasic (apurinic/apyrimidinic, AP) sites on complementary oligodeoxynucleotides. Use of the symmetrical triamine, tris(2-aminoethyl)amine, in this reaction sequence enabled preparation of a cross-linked DNA duplex bearing a derivatizable aminoethyl group.

The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-...

Apurinic/apyrimidinic endonuclease 1 (APE1) exonuclease plays a vital proofreading role in the base excision repair pathway by removing 3’end group, including oxidatively damaged DNA bases, chain terminating nucleotide analog drugs, terminal blocking groups and mismatched bases. Herein, a simple and real time fluorometric method was developed for detecting APE1 exonuclease activity using molecular beacon as substrate. Experimental results demonstrated that the detection limit of this method was 0.01 U/μL for APE1, and the Km value was (0.27 ± 0.10) μmol/L. Moreover, the method was used to screen antibiotics and the results showed that streptomycin sulfate and gentamycin sulfate had a significant inhibitory effect on APE1 exonuclease activity in a concentration-dependent manner. Meanwhile, the assay was used for investigating the inhibitory effects of heavy metal ions on APE1 exonuclease activity. Finally, the method was further used to monitor the effect of heavy metal ions treatment of the APE1 exonuclease activity of ramie, and the results indicated that APE1 exonuclease activity was inhibited in a concentration-dependent manner after treatment with Pb2+ and Cd2+, which was consistent with heavy metal ions assay in vitro. In summary, this method provided an alternative tool for the biochemical analysis for APE1 exonuclease activity, which was hopeful for the assessment for heavy metal ions adsorption according to the enzymatic activity change in phytoremediation.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics., Graphical Abstract Graphical AbstractMechanism of DNA sequence recognition and cleavage by L1-EN endonuclease domain during initiation of the LINE-1 retrotransposon target-primed reverse transcription.

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.

Ionizing radiation damage to DNA plays a fundamental role in cancer therapy. X-ray photoelectron-spectroscopy (XPS) allows simultaneous irradiation and damage monitoring. Although water radiolysis is essential for radiation damage, all previous XPS studies were performed in vacuum. Here we present near-ambient-pressure XPS experiments to directly measure DNA damage under water atmosphere. They permit in-situ monitoring of the effects of radicals on fully hydrated double-stranded DNA. The results allow us to distinguish direct damage, by photons and secondary low-energy electrons (LEE), from damage by hydroxyl radicals or hydration induced modifications of damage pathways. The exposure of dry DNA to x-rays leads to strand-breaks at the sugar-phosphate backbone, while deoxyribose and nucleobases are less affected. In contrast, a strong increase of DNA damage is observed in water, where OH-radicals are produced. In consequence, base damage and base release become predominant, even though the number of strand-breaks increases further. Ionizing radiation damage to DNA plays a fundamental role in cancer therapy, whereby water radiolysis effects are important to take into account. Here, near-ambient-pressure X-ray photoelectron-spectroscopy is used to directly measure DNA damage during irradiation in water and compared to that in a dry atmosphere.

One of the most common DNA lesions is the appearance of apurinic/apyrimidinic (AP-) sites. The main repair pathway for AP sites is initiated by apurinic/apyrimidinic endonuclease 1 (APE1). Upon hydrolysis of the phosphodiester bond by this enzyme, a one nucleotide gap flanked by 3′-hydroxyl and 5′‑deoxyribose phosphate groups on the 5′-side of the AP site is formed. After hydrolysis of the AP site, APE1 remains associated with the product for some time. In the present work, the ability of APE1 to form a product of covalent attachment of APE1 to DNA containing a gap with a 5′-deoxyribose phosphate residue was demonstrated. In addition, it was found that while in a complex with the product of hydrolysis of the AP site, APE1 exhibits 5'‑deoxyribose phosphate lyase activity, cleaving off the 5′-deoxyribose phosphate residue. The presence of lyase activity in APE1 may be important for the repair of AP sites if there is a deficiency of, or mutations in DNA polymerase β, the main enzyme that removes the 5′-deoxyribose phosphate group.

G-quadruplex-based complexes have been widely used in various analytical methods due to their outstanding capabilities of generating colorimetric, fluorescent or electrochemical signals. However, since loop sequences in traditional G-quadruplex structures are quite short, it is difficult to establish biosensors solely using G-quadruplex-based complexes. Herein, we attempted to lengthen the loop sequences of G-quadruplex structures and found that G-quadruplex-hemin DNAzymes (G-DNAzymes) with long loops (even 30 nucleotides) maintain high peroxidase activity. In addition, the peroxidase activity is not affected by the hybridization of the long loop with its complementary counterpart. Consequently, G-DNAzyme can be endowed with an additional function by taking the long loop as a recognition element, which may facilitate the construction of diverse colorimetric biosensors. Furthermore, by designing an apurinic/apyrimidinic site or a complementary sequence of microRNA-21 (miRNA-21) in long loops, bifunctional G-DNAzymes can be split in the presence of apurinic/apyrimidinic endonuclease 1 (APE1) or miRNA-21, decreasing their peroxidase activities. Accordingly, APE1 and miRNA-21 are quantified using 3,3',5,5'-tetramethylbenzidine as a chromophore. Using the G-DNAzyme, APE1 can be detected in a linear range from 2.5 to 22.5 U mL-1 with a LOD of 1.8 U mL-1. It is to be noted that benefitting from duplex-specific nuclease-induced signal amplification, the linear range of the miRNA-21 biosensor is broadened to 5 orders of magnitude, while the limit of detection is as low as 73 fM. This work demonstrates that G-DNAzymes with long loops can both generate signals and recognize targets, providing an alternative strategy to design G-quadruplex-based analytical methods.

The simultaneous detection of multiple microRNAs (miRNAs) will facilitate early clinical diagnosis. Herein, we demonstrate the integration of multicolor fluorophore-encoded cascade signal amplification with single-molecule detection for simultaneous measurement of different miRNAs in lung cancer tissues. This assay involves two linear templates and two circular templates without the requirement of any fluorescent-labeled probes. The binding of target miRNAs to their corresponding linear templates initiates the cyclic strand displacement amplification, generating many triggers which can specifically hybridize with the corresponding biotin-labeled AP probes to initiate the apurinic/apyrimidic endonuclease 1-assisted cyclic cleavage reaction for the production of more biotin-labeled primers for each miRNA. The resultant two primers can react with their corresponding circular templates to initiate rolling circle amplification which enables the incorporation of Cy5-dCTP/Cy3-dGTP nucleotides, resulting in the simultaneous production of abundant biotin-/multiple Cy5/Cy3-labeled DNA products. After magnetic separation and exonuclease cleavage, the amplified products release abundant Cy5 and Cy3 fluorescent molecules which can be simply monitored by single-molecule detection, with Cy3 indicating miR-21 and Cy5 indicating miR-155. This assay involves three consecutive amplification reactions, enabling the conversion of extremely low abundant target miRNAs into large numbers of Cy5/Cy3 fluorophore-encoded DNA products which can release abundant fluorescent molecules for the generation of amplified signals. This assay exhibits high sensitivity, good selectivity, and the capability of multiplexed assay. This method can simultaneously quantify miR-155 and miR-21 in living cells and in lung cancer tissues, and it can distinguish the expression of miRNAs between non-small cell lung cancer patients and healthy persons. The accuracy and reliability of the proposed method are further validated by quantitative reverse transcription polymerase chain reaction., We integrate single-molecule detection with multicolor fluorophores-encoded cascade signal amplification for simultaneous detection of different miRNAs in lung cancer tissues.

Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1/Ref-1, also called APE1) is a multifunctional enzyme with crucial roles in DNA repair and reduction/oxidation (redox) signaling. APE1 was originally described as an endonuclease in the Base Excision Repair (BER) pathway. Further study revealed it to be a redox signaling hub regulating critical transcription factors (TFs). Although a significant amount of focus has been on the role of APE1 in cancer, recent findings support APE1 as a target in other indications, including ocular diseases [diabetic retinopathy (DR), diabetic macular edema (DME), and age-related macular degeneration (AMD)], inflammatory bowel disease (IBD) and others, where APE1 regulation of crucial TFs impacts important pathways in these diseases. The central responsibilities of APE1 in DNA repair and redox signaling make it an attractive therapeutic target for cancer and other diseases.

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Apurinic/apyrimidinic (AP) endonucleases are vital DNA repair enzymes, and proposed to be a prognostic biomarker for various types of cancer in humans. Numerous DNA sensors have been developed to evaluate the extent of nuclease activity but their DNA termini are not protected against other nucleases, hampering accurate quantification. Here we developed a new fluorescence enhancement (FE)-based method as an enzyme-specific DNA biosensor with nuclease-protection by three functional units (an AP-site, Cy3 and termini that are protected from exonucleolytic cleavage). A robust FE signal arises from the fluorescent cis–trans isomerization of a cyanine dye (e.g., Cy3) upon the enzyme-triggered structural change from double-stranded (ds)DNA to single-stranded (ss)DNA that carries Cy3. The FE-based assay reveals a linear dependency on sub-nanomolar concentrations as low as 10−11 M for the target enzyme and can be also utilized as a sensitive readout of other nuclease activities.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and β configurations, via an electrophilic aldehyde intermediate...

A clustered DNA damage site (cluster), in which two or more lesions exist within a few helical turns, is believed to be a key factor determining the fate of a living cell exposed to a DNA damaging agent such as ionizing radiation. However, the structural details of a cluster such as the number of included lesions and their proximity are unknown. Herein, we develop a method to characterize a cluster by fluorescence anisotropy measurements based on Forster resonance energy transfer (homo-FRET). Plasmid DNA (pUC19) was irradiated with 2.0 and 0.52 MeV/u 4He2+, or 0.37 MeV/u 12C5+ ion beams (linear energy transfer: ~ 70, ~ 150, ~ 760 keV/μm, respectively) and 60Co γ-rays as a standard (~ 0.2 keV/μm) in the solid state. The irradiated DNA was labeled with an aminooxyl fluorophore (Alexa Fluor 488) to the aldehyde/ketone moieties such as apurinic/apyrimidinic sites. Homo-FRET analyses provided the apparent base separation values between lesions in a cluster produced by each ion beam track as 21.1, 19.4, and 18.7 base pairs. The production frequency of a cluster increases with increasing linear energy transfer of radiation. Our results demonstrate that homo-FRET analysis has the potential to discover the qualitative and the quantitative differences of the clusters produced not only by a variety of ionizing radiation but also by other DNA damaging agents.

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.

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N(7)POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 10(8) nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and non-smokers. Rats treated with 5 or 21 mg/kg bw NNK for four days by s.c. injection had 2–6 and 2–17 times more AP sites than controls in liver and lung DNA (p

Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyzethe phosphodiester bond on the 5-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidinepurine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.

International audience; Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88 °C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95 °C for 1.0 h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0–9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3′-5′ exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.

Molecular recognition of proteins is critical for study and manipulation of protein-related biological processes. However, design and synthesis of abiotic receptors for precise recognition of proteins still remains a challenging task. Herein, we developed a universal sequential surface-imprinting strategy that integrated two different types of imprinting reactions to construct artificial protein receptors with high selectivity. Employing dopamine self-polymerization and boronate/diol complexation as the first-step and second-step imprinting reactions, respectively, we synthesized surface-imprinted magnetic nanocomposites against two different enzyme proteins: deoxyribonuclease I (DNase I) and apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). The obtained nanocomposites both showed strong and specific binding toward their respective template proteins. Moreover, the bound enzymes could be totally recovered with high activity under mild buffer conditions. These antibody-like specific and reversible binding properties enabled effective purification and enrichment of the low-abundance target proteins from complex serum samples. Compared to existing one-pot or one-step imprinting methods, the proposed sequential surface-imprinting approach offers a more flexible combination of different functional monomers and greatly enhances the performance and biocompatibility of the imprinted materials. The generality and simplicity of the sequential imprinting strategy would make it an appealing and competitive method to prepare artificial protein receptors.

Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions., REV7 has emerged as a critical regulator of DNA double-strand breaks repair. Here, the authors show that REV7 is crucial for both antibody class switch recombination and somatic hypermutation in activated B cells, in addition to their survival upon AID-deamination.

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5’ DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5’ terminus stimulates resection by EXO1. Ribonucleotides within a 5’ flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases., DNA double-strand break repair by homologous recombination initiates with nucleolytic resection of the 5’ DNA strand at the break ends. Here, the authors reveal that the lesion context influences the action and efficiency of the long range resection factors EXO1 and BLM-DNA2.

Thymine:guanine base pairs are major promutagenic mismatches occurring in DNA metabolism. If left unrepaired, these mispairs can cause C to T transition mutations. In humans, T:G mismatches are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (hMBD4) and thymine-DNA glycosylase. Unlike lesion-specific DNA glycosylases, T:G-mismatch-specific DNA glycosylases specifically recognize both bases of the mismatch and remove the thymine but only from mispairs with guanine. Despite the advances in biochemical and structural characterizations of hMBD4, the catalytic mechanism of hMBD4 remains elusive. Herein, we report two structures of hMBD4 processing T:G-mismatched DNA. A high-resolution crystal structure of Asp560Asn hMBD4-T:G complex suggests that hMBD4-mediated glycosidic bond cleavage occurs via a general base catalysis mechanism assisted by Asp560. A structure of wild-type hMBD4 encountering T:G-containing DNA shows the generation of an apurinic/apyrimidinic (AP) site bearing the C1′-(S)-OH. The inversion of the stereochemistry at the C1′ of the AP-site indicates that a nucleophilic water molecule approaches from the back of the thymine substrate, suggesting a bimolecular displacement mechanism (SN2) for hMBD4-catalyzed thymine excision. The AP-site is stabilized by an extensive hydrogen bond network in the MBD4 catalytic site, highlighting the role of MBD4 in protecting the genotoxic AP-site.

Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The mono-alkylated N7-guanine (NM-G) adduct, and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened N(5)-substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM–Fapy-G adducts in liver, lung, and spleen over 168 hours. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 hours post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing in vivo. The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.

Base excision DNA repair (BER) is an important process used by all living organisms to remove nonbulky lesions from DNA. BER is usually initiated by DNA glycosylases that excise a damaged base leaving an apurinic/apyrimidinic (AP) site, and an AP endonuclease then cuts DNA at the AP site, and the repair is completed by correct nucleotide insertion, end processing, and nick ligation. It has emerged recently that the BER machinery, in addition to genome protection, is crucial for active epigenetic demethylation in the vertebrates. This pathway is initiated by TET dioxygenases that oxidize the regulatory 5-methylcytosine, and the oxidation products are treated as substrates for BER. T:G mismatch-specific thymine-DNA glycosylase (TDG) and AP endonuclease 1 (APE1) catalyze the first two steps in BER-dependent active demethylation. In addition to the well-structured catalytic domains, these enzymes possess long tails that are structurally uncharacterized but involved in multiple interactions and regulatory functions. In this review, we describe the known roles of the tails in TDG and APE1, discuss the importance of order and disorder in their structure, and consider the evolutionary aspects of these accessory protein regions. We also propose that the tails may be important for the enzymes' oligomerization on DNA, an aspect of their function that only recently gained attention.

Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time in vitro formation of the ternary XRCC1 complex with the key enzymes of SP BER - DNA polymerase β (Polβ) and DNA ligase IIIα (LigIIIα) - using the fluorescence-based technique. It was found that Polβ directly interacts with LigIIIα, but their complex is less stable than the XRCC1-Polβ and XRCC1-LigIIIα complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polβ), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.

Strand displacement DNA synthesis is essential for DNA replication. Gp90, the sole DNA polymerase of Pseudomonas aeruginosa phage 1, can bypass multiply DNA lesions. However, whether it can perform strand displacement synthesis is still unknown. In this work, we found that gp90 exo- could perform strand displacement synthesis, albeit its activity and processivity were lower than those of primer extension. Gp90 exo- itself could not unwind Y-shaped or fork DNA. Tail and gap at DNA fork were necessary for efficient synthesis. High GC content obviously inhibited strand displacement synthesis. Consecutive GC sequence at the entrance of fork showed more inhibition effect on DNA synthesis than that in the downstream DNA fork. The fraction of productive polymerase and DNA complex (A values) was higher for fork than gap; while their average extension rates (kp values) were similar. However, both A and kp values were lower than those for the primer/template (P/T) substrate. The binding of gp90 exo- to fork was tighter than P/T or gap in the absence of dATP. In the presence of dATP to form ternary complex, the binding affinity of gp90 exo- to P/T or gap was increased compared with that in the binary complex. Abasic site, 8-oxoG, and O6-MeG inhibited and even blocked strand displacement synthesis. This work shows that gp90 exo- could perform strand displacement DNA synthesis at DNA fork, discovering the presence of new functions of PaP1 DNA polymerase in DNA replication and propagation of PaP1.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C‐terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C‐terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild‐type fish. There was no significant difference in the frequency of DENA‐induced mutations between mutant with reduced dCMP transferase activity and wild‐type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH., LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. We found defect in the catalytic activity of rev1 exhibited a higher tumorigenic rate and high frequency of LOH. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

Here we report that incorporation of an abasic site to DNA minidumbbells formed by natural sequences can lead to significant enhancements in their thermodynamic stability. Based on these stable minidumbbells, the first metal ion-controlled molecular switch which can regulate instant and reversible DNA duplex formation and dissociation has been constructed.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential multifunctional protein in mammals involved in base excision DNA repair (BER), regulation of gene expression and RNA metabolism. Its major enzymatic function is incision of AP sites. Poly(ADP-ribose) polymerase 1 (PARP1) modifies itself and target proteins with poly(ADP-ribose) (PAR), contributing to regulation of many processes. To understand molecular basis of functional cooperation between APE1 and PARP1 in BER, we examined PAR-binding activity and ADP-ribosylation of human APE1 in comparison with known targets of PARP1, using the full-length, N-terminally truncated and catalytically inactive forms of APE1. The protein binds preferentially large ADP-ribose polymers, being very similar to DNA polymerase β (Polβ) but contrasting with the scaffold XRCC1 protein. The interaction with PAR involves the universally conserved catalytic portion and the eukaryote-specific extension of APE1. The ADP-ribosylation of APE1 depends on the structure of PARP1-activating DNA, contrasting APE1 with Polβ and XRCC1. Relative levels of APE1 modification in the presence of different DNA substrates were found to correlate with affinities of the DNAs for APE1 and substrate activities in the enzymatic incision, suggesting the ADP-ribosylation to occur within the DNA-mediated ternary complex. This conclusion was confirmed by importance of the length of DNA region 3' to the AP site for the modification. Deletion of the N-terminal extension of APE1 produced no significant influence on both the ADP-ribosylation efficiency and hydrolytic stability of the modified protein, suggesting localization of target amino acids in the conserved catalytic portion. The most efficient ADP-ribosylation of the catalytically inactive APE1 mutant was shown to reduce the level of PARP1 automodification, suggesting possible role of APE1 in modulating PARP1 activity. Our data on primary role of DNA in controlling the PARP-catalysed modification provide new insights into mechanisms of protein targeting for ADP-ribosylation.

DNA double-strand breaks and chromosomal aberrations after treatment with N-alkylating agents likely arise as a result of replication fork collision with single-strand breaks generated during base excision repair., Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER.

Nuclear, mitochondrial and plastidic DNA is constantly exposed to conditions, such as ultraviolet radiation or reactive oxygen species, which will induce chemical modifications to the nucleotides. Unless repaired, these modifications can lead to mutations, so the nucleus, mitochondria and plastids each contains a number of DNA repair systems. We here describe assays for measuring the enzyme activities associated with the base-excision repair pathway in potato tuber mitochondria. As the name implies, this pathway involves removing a modified base and replacing it with an undamaged base. Activity of each of the enzymes involved, DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase and DNA ligase can be measured by incubating a mitochondrial extract with a specifically designed oligonucleotide. After incubation, the reaction mixture is separated on a polyacrylamide gel, and the amounts of specific products formed is estimated by autoradiography, which makes it possible to calculate the enzymatic activity.

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3’ to 5’ exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3’-end of a DNA strand during replication or repair. Importantly, 3’-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3’-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

The 5′,8-cyclo-2′-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered—the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5′-end side of cdPu than on its 3′-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.