DNA Repair

DNA polymerases are important for maintaining genomic stability by protecting against mutagenic lesions caused by external and internal factors. If left unrepaired, DNA damage can lead to replication errors resulting in changes in DNA sequences that could impact peptide sequences and gene expression, as well as lead to chromosomal breaks. Highlighting the importance of DNA polymerase repair function, variant DNA polymerases and cofactors of DNA repair pathways have been identified in many different cancer types. Recently, variant forms of DNA polymerase Q (POLQ) have been identified in patient isolated melanoma tumors. Previous work identifying biochemical characteristics of these variants has shown that they display aberrant DNA polymerase activity compared to wild-type (WT). To better understand the role these variants have in DNA repair, genomic stability and cell survival, we tested their ability to bypass and extend DNA past cyclobutane pyrimidine dimers (CPD) as well as prevent cell death when exposed to ultra-violet (UV) radiation. Biochemically we show that the patient derived variants of POLQ tested here display decreased efficiency during DNA bypass and extension of CPD lesions and prefer to incorporate purines. In addition, two of the three variants protect against UV induced cell death. Together these data suggest that POLQ variants can support cell survival and further supports that POLQ variants can act as both protectors of cell viability as well as drivers of genomic instability, characteristics important in cancer cells. HIGHLIGHTS[vrecto] POLQ variants have decreased efficiency during bypass and extension of CPD damaged DNA compared to WT POLQ [vrecto]POLQ is able to bypass and extend cyclobutane pyrimidine dimers, but prefers purine over pyrimidine incorporation [vrecto]POLQ variants can protect against UV induced cell death.

DNA repair pathways are frequently defective in human cancers. DNA double strand breaks (DSBs) are most often repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ). Alterations in repair pathways can indicate sensitivity to therapeutic agents such as PARP inhibitors, cisplatin, and immunotherapy. Thus, functional assays to measure rates of HR and NHEJ are of significant interest. Several methods have been developed to measure rates of HR or NHEJ; however, there is a need for functional cell-based assays that can measure rates by both major DNA DSB pathways simultaneously. Here, we describe the RepairSwitch assay, a flow cytometry assay to assess rates of HR and NHEJ mediated repair of Cas9 programmed DSB simultaneously using a novel fluorescence switching reporter system. The assay exhibits low background signal and is capable of detecting rare repair events in the 1 in 10,000 range. We demonstrate the utility of RepairSwitch by measuring the potency of inhibitors of ATM (KU-60019, KU-55933), DNA-PK (NU7441), and PARP (Olaparib) on modulating DSB repair rates in HEK293FT cells. The selective ATM inhibitor KU-60019 inhibited HR rates with IC50 of 915 nM. Interestingly, KU-60019 exposure led to a dose responsive increase in rates of NHEJ. In contrast, the less selective ATM inhibitor KU-55933, which also has activity on DNA-PK, showed inhibition of both HR and NHEJ. The selective DNA-PK inhibitor NU7441 inhibited NHEJ efficiency with an IC50 of 299 nM, and showed a dose responsive increase in HR. The PARP inhibitor Olaparib showed lower potency in modulating HR and NHEJ. We next used the RepairSwitch assay to assess how pharmacological and genetic inhibition of DNA methyltransferases (DNMT) impacted rates of HR and NHEJ. The DNMT inhibitor decitabine reduced HR, but increased rates of NHEJ, both in a dose responsive manner, in both HEK293FT and HCT116 cells (IC50 for HR of 187 nM and 1.4 uM respectively). Knockout of DNMT1 and DNMT3B increased NHEJ, while knockout of DNMT3B, but not DNMT1, reduced HR. These results illustrate the utility of RepairSwitch as a functional assay for measuring changes in rates of DSB repair induced by pharmacological or genetic perturbation. Furthermore, the findings illustrate the potential for one DNA repair mechanism to compensate in part for loss of another. Finally, we showed that inhibition of DNMT can lead to reduction of HR and increase in NHEJ, providing some additional insight into recently observed synergy of DNMT inhibitors with PARP inhibitors for cancer treatment.

The analysis of DNA repair mechanisms is of fundamental importance to understand how cells remove DNA damage and maintain their genome stability. Investigating the dynamic association of proteins at sites of active DNA synthesis has been successfully performed at DNA replication forks, providing important information on the process, and allowing the identification of new players acting at these sites. However, the applicability of these studies to DNA repair events at sites of nascent unscheduled DNA synthesis (UDS) in non-proliferating cells has been never tested. Here, we describe the analysis of dynamics association of protein participating in nucleotide excision repair (NER), and in other DNA repair processes, at sites of nascent UDS in non-proliferating cells, to avoid interference by DNA replication. Labeling with 5-ethynyl-2-deoxyuridine (EdU) after DNA damage, followed by click reaction to biotinylate these sites, permits the analysis of dynamic association of proteins, such as DNA polymerases {delta} and {kappa}, as well as PCNA, to active DNA repair synthesis sites. The suitability of this technique to identify new factors present at active UDS sites is illustrated by two examples of proteins previously unknown to participate in the UV-induced DNA repair process.

Numerical abnormalities in chromosomal states, referred to as aneuploidy, is commonly observed in many cancer cells. Although numerous internal and external factors induce aneuploidy, the primary cause of aneuploidy in humans remains unclear. DNA damage is identified as a potential cause of aneuploidy by inducing chromosome segregation errors. However, a direct relationship between DNA damage and aneuploidy remains poorly understood. A major reason for this is the extremely low frequency of aneuploidy in cultured cells, making quantitative analyses challenging. In this study, we investigated the relationship between DNA damage and aneuploidy in cell lines containing minichromosomes. These chromosomes are more prone to loss than normal chromosomes, with the rate of loss substantially increased following exposure to various DNA-damaging agents. To determine whether damaged chromosomes were subjected to direct loss or whether chromosome loss occurred as an indirect consequence of a prolonged G2 phase or other factors, we used the CRISPR-Cas9 system to introduce a single DNA double-strand break (DSB) on a minichromosome. The rate of minichromosome loss increased by approximately seven-fold compared with that of the control. Furthermore, the loss rate was significantly elevated in the absence of KU70, a key factor in non-homologous end joining, and upon inhibition of ataxia telangiectasia mutated (ATM), a DNA damage checkpoint protein. Finally, two closely spaced nicks, believed to generate a 5-overhang, were also shown to induce minichromosome loss. These findings indicated that a single DSB or two closely spaced nicks can cause aneuploidy if improperly repaired in vertebrates.

A DNA double strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination (HR) or non-homologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of HR, is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harbouring nuclease dead dna2-1 compared to dna2{Delta} pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harbouring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater defect in resection compared to cells where DNA2 was deleted. Both the decreased level of resection and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change, however Nej1 increased as did the frequency of alt-EJ/ MMEJ repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/564088v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c8280borg.highwire.dtl.DTLVardef@1bcf245org.highwire.dtl.DTLVardef@1c59f43org.highwire.dtl.DTLVardef@15b18d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Breast Cancer Susceptibility Gene 1 (BRCA1) codes for a DNA repair protein that facilitates the repair of double-stranded DNA breaks (DSBs) in human cells through the homologous recombination (HR) pathway. Mutations of BRCA1 are highly associated with breast cancer; however, many variants remain unclassified with unknown cellular phenotypes. The DNA binding activity of BRCA1 is localized primarily to its central region, which can be divided into two distinct domains: DNA Binding Domain 1 (DBD1; amino acids (aa) 330-554) and 2 (DBD2; aa 894-1057). We previously proposed a model in which DBD1 targets BRCA1 to DSBs for the promotion of DNA end resection, while DBD2 targets BRCA1 to telomeres to function in chromatin remodeling and telomere regulation. In this study, we hypothesized that unknown DBD variants (T374I, K408E, N417S, N909I, M1008I, and R1028H) with similar properties to known disease-causing variants (Q356H, F461L, R496H, D940Y, S1027N, and E1038G) would also be pathogenic. The affinities of each variant for single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and a G-quadruplex (G4) sequence were measured via biolayer interferometry. The DNA repair phenotypes of each variant were analyzed by overexpression in HEK cells to determine correlation between binding activity and DNA damage response. Altogether, these results provide insight into how missense mutations affect the ability of BRCA1 DBDs to facilitate the DNA damage response.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically Rad51, have prominent roles in the S phase. Using different cellular models, Rad51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of Rad51 in the S phase. Rad51 inhibition in non-transformed cell lines did not have a major effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, Rad51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of Rad51 did not compromise fork integrity when subjected to hydroxyurea treatment. Rad51 inhibition also did not decrease the ability to restart, but rather compromised, fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, Rad51 inhibition reduced replication fork speed in these cells and increased {gamma}H2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase to chromatin, as observed when inhibiting Rad51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on Rad51 activity under basal conditions to design new therapies that specifically target cancer cells.

Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical non-homologous end-joining pathway, but it may have roles in modulating gene expression and thus, the overall cellular response to DNA damage. Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Downregulation of pathways involved in biosynthesis were observed in both genotypes, but upregulated biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/{beta}-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trial as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

Mitochondria lack nucleotide excision repair; however, mitochondrial DNA (mtDNA) is resistant to mutation accumulation following DNA damage. These observations suggest additional damage sensing or protection mechanisms. Transcription Factor A, Mitochondrial (TFAM) compacts mtDNA into nucleoids and binds differentially to certain forms of DNA damage. As such, TFAM has emerged as a candidate for protecting mtDNA or sensing damage. To examine the possibilities that TFAM might protect DNA from damage or act as a damage sensing protein for irreparable forms of mtDNA damage, we used live-cell imaging and HeLa cell-based assays, atomic force microscopy (AFM), and high-throughput protein-DNA binding assays to characterize the binding properties of human TFAM to ultraviolet-C (UVC) irradiated DNA and the cellular consequences of UVC irradiation. Our cell data show increased TFAM mRNA after exposure and suggest an increase in mtDNA degradation without a loss in mitochondrial membrane potential that might trigger mitophagy. Our protein-DNA binding assays indicate a reduction in sequence specificity of TFAM following UVC irradiation and a redistribution of TFAM binding throughout the mitochondrial genome. Our AFM data show increased compaction of DNA by TFAM in the presence of damage. Despite the TFAM-mediated compaction of mtDNA in vitro, we do not observe any protective effect of increased TFAM protein on DNA damage formation in cells or in vitro. Increased TFAM protein did not alter levels of mtDNA damage over time after UVC exposure in vivo, but knockdown of TFAM did alter mtDNA damage levels in HeLa cells both at baseline and after UVC exposure. Taken together, these studies indicate that UVC-induced DNA damage alters TFAM binding and promotes compaction by TFAM in vitro. We hypothesize that that TFAM may act as a damage sensing protein in vivo, sequestering damaged genomes to prevent mutagenesis by facilitating removal or suppression of replication.

In eukaryotes, mismatch repair begins with MutS homolog (MSH) complexes, which scan newly replicated DNA for mismatches. Upon mismatch detection, MSH complexes recruit the PCNA- stimulated endonuclease Mlh1-Pms1/PMS2 (yeast/human), which nicks the DNA to allow downstream proteins to remove the mismatch. Past work has shown that although Mlh1-Pms1 is an ATPase and this activity is important in vivo, ATP is not required to nick DNA. Our data, using yeast as a model, suggests that Mlh1-Pms1 forms oligomeric complexes that drive DNA conformational rearrangements using the proteins ATPase activity. Experiments with non-B-form DNA structures, common in microsatellite regions, show that these structures inhibit Mlh1-Pms1s activities, likely through impeding Mlh1-Pms1-dependent DNA conformational changes. This could explain an additional mode for instability in these regions of the genome. These findings highlight the importance of DNA compaction and topological rearrangements in Mlh1-Pms1s function and provide insight into how mismatch repair relies on DNA structure to coordinate events. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=31 SRC="FIGDIR/small/633381v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@12a2bcdorg.highwire.dtl.DTLVardef@1a1a053org.highwire.dtl.DTLVardef@24e681org.highwire.dtl.DTLVardef@99297c_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA polymerase {zeta} (Pol {zeta}) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases {eta}, {zeta}, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol {zeta} complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol {zeta} alone or Pol {eta}. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol {eta}, Pol {zeta}, and Rev1-Pol {zeta}. Rev1-Pol {zeta}, and particularly Pol {zeta} alone showed a tendency to stall before the ICL, whereas Pol {eta} stalled just after insertion across the ICL. The stalling of Pol {eta} directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol {eta} providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol {zeta} directly past the ICL was observed, suggesting that the proposed function of Pol {zeta} as an extender DNA polymerase is also required for ICL repair.

Extrachromosomal circular DNAs (eccDNA) are widespread in normal and cancer cells and are known to amplify oncogenic genes. However, the mechanisms that form eccDNA have never been fully elucidated due to the complex interactions of DNA repair pathways and lack of a method to quantify eccDNA abundance. Through the development of a sensitive and quantitative assay for eccDNA we show that the formation of eccDNA is through resection dependent repair of double-strand DNA breaks, especially micro-homology mediated end joining, and through mismatch repair. The most significant decreases in eccDNA levels occurred in cells lacking PARP1, POLQ, NBS1, RAD54, and FAN1. Further, a significant increase in eccDNA occurred in cells lacking c-NHEJ proteins DNA-PKcs, XRCC4, XLF, LIG4 and 53BP1. This suggests that when alt-NHEJ pathways are utilized to repair DNA breaks by necessity, the formation of eccDNA is increased. Induced and site-directed double-strand DNA breaks increase eccDNA formation, even from a single break. Additionally, we find that eccDNA levels accumulate as cells undergo replication in S-phase and that levels of eccDNA are decreased if DNA synthesis is prevented. Together, these results show that the bulk of eccDNA form by resection based alt-NHEJ pathways, especially during DNA replication and the repair of double-strand breaks.

Topoisomerase II is a nuclear enzyme needed for dealing with topological entanglements in the DNA arising from replication and transcription. The N-terminal region and core of the protein are utilized in the catalytic cycle of the enzyme, which generates a transient double-stranded break in one segment of DNA and passes another segment through the break. The C-terminal domain is a large, intrinsically disordered region that appears to be involved in regulating the function of the enzyme both in terms of substrate selection and the level of activity of the enzyme. In a previous study, we explored eleven targeted mutations to the C-terminal domain. This present study explores six of these mutants to determine whether there are any defects in closure of the N-terminal clamp and whether an experimental compound known as a Cu(II)-thiosemicarbazone affects DNA cleavage with the mutants. Based upon our results, the mutants are able to close the N-terminal clamp, but some of the mutants that displayed the least clamp closing activity also had the lowest catalytic activity. Further, Cu-APY-ETSC did impact the ability of the enzymes to cleave DNA to similar levels as seen with the WT enzyme. These results lay the groundwork for additional analyses of the C-terminal domain and indicate the C-terminal domain regions tested did not influence the action of Cu-APY-ETSC except at the level of coordination between the two active sites.

The human RAD52 protein is thought to have multiple roles in the mechanisms of repairing DNA double-strand breaks that are caused by replication errors and reactive oxygen species. One such role is to mediate the formation of a displacement loop (D-loop), which is a critical reaction intermediate in homologous recombinational repair. RAD52 is suggested to promote the formation of D-loops when facilitating DNA synthesis at stalled or collapsed replication forks during mitosis. However, RAD52-mediated D-loop formation remains poorly characterized, and the detailed molecular mechanism of the D-loop formation reaction catalyzed by RAD52 is still unclear. In the present study, we developed a gel-based assay that enables rapid detection of RAD52-mediated D-loop formation. This assay utilizes a fluorophore-labeled, single-stranded DNA substrate. In addition to the rapid detection of D-loops, D-loop extension was observed when DNA polymerase was added to the reaction. This assay can also be used for screening large numbers of compounds that either stimulate or inhibit RAD52-mediated D-loop formation. The D-loop formation assay developed in this study is potentially useful for mechanistic studies of DSB repair involving RAD52-mediated D-loop formation, as well as for screening compounds with potential therapeutic effects.

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased [~]2-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol {beta} performs multiple insertions on G4 substates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol {beta} products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol {beta} prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

PriA is a member of the SuperFamily 2 helicase family. Its role in vivo is to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the E.coli protein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3-OH placed at the end of the nascent leading strand at the fork. When both the 3-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

Although mismatch repair (MMR) defects are associated with high risk of malignancy, the specific oncogenic drivers pertinent to MMR-affected cancers are poorly characterized. The heterozygous ATR-I774Yfs*5 mutation, the result of strand slippage in a poly-A tract of the Ataxia Telangiectasia and Rad3 related (ATR) gene, is overexpressed in MMR-defective malignancies including colorectal carcinoma (CRC) and is the most common ATR mutation in cancer. Here, we explore the contribution of ATR-I774Yfs*5 to genomic integrity. Using heterozygous ATR-I774Yfs*5 HCT-116 cells to mimic the native mutation, we found this mutation reduced ATR activity as measured by damage-induced Chk1 phosphorylation at S317 and ATR autophosphorylation ATR at T1989. ATR-I774Yfs*5 expression impaired genomic stability as visualized by the appearance of micronuclei in two stable expression models as well as in cell lines transfected with ATR-I774Yfs*5. Micronucleus development was dependent on replication and independent of ATR copy number. ATR-I774Yfs*5 expression did not alter cellular viability, cell cycle progression, or replicative rate, suggesting this mutation is well-tolerated despite its destabilizing effect on the genome. Taken together, these data suggest that the ATR-I774Yfs*5, whose development is favored in the context of MMR deficiency, may represent an important driver of a mutator phenotype by promoting genomic instability.

Formation of a repair enzyme complex is beneficial to DNA repair. Despite the fact that mitochondrial base excision repair (mtBER) enzymes DNA polymerase gamma (Pol {gamma}) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex, the functional role of the interaction in mtBER has not been characterized. We report studies that PARP1 regulates Pol {gamma} activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol {gamma}, while increasing NAD+ level to physiological concentration enables Pol {gamma} to resume maximum repair activity. Pol {gamma} is PARylated when bound to DNA repair intermediates, and PARylation is essential for Pol {gamma} repair activity. The PARP1 inhibitor Olaparib that abolishes PARP1 catalytic activity suppresses Pol {gamma} gap-filling synthesis at physiological concentrations of NAD+, suggesting inhibiting PARP1 activity would increase mtDNA mutations. Because NAD+ cellular levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is correlated with cellular metabolic state and integrity of the respiratory chain. Our results revealed a molecular basis of drug toxicity from prolonged usage of PARP1 inhibitors in treating cardiac dysfunctions

DNA ligase (LIG) 1 and LIG3 repair broken single-strand breaks in the phosphodiester backbone at the final ligation step of DNA excision repair pathways, and complement each other during nuclear replication in case of unligated Okazaki fragments. We previously reported that both ligases discriminate against nicks containing non-canonical ends and ligate gap intermediate if left unfilled by DNA polymerases. However, it remains unknown how the dynamics of DNA binding differ for gap versus nick substrates by LIG1 and LIG3 at single-molecule level. Here, using total internal reflection fluorescence (TIRF) and ligation assays, we showed that LIG3 binds less frequently but forms longer-lived complex than LIG1 for nicks containing canonical A:T, mismatch G:T, and damaged 8oxoG:A, and they exhibit subtle differences in discriminating unusual ends. Moreover, our results identified gap DNA as a new target to which LIG1 and LIG3 can bind as efficient as their preferential nick sites. We showed gap ligation and observed that more percentage of LIG1 molecules form stable long-lived complex on DNA containing one nucleotide gap, whereas LIG3 forms short-lived gap complex without any differences in the percentage of molecule forming gap-bound complex. Finally, our findings demonstrated that LIG1 can still stably bind to larger gaps with better recognition, whereas LIG3 binding becomes further infrequent and shorter-lived. Overall, our study provides single-molecule insights into intricate differences between LIG1 and LIG3 for binding to a range of mutagenic and deleterious DNA repair and replication intermediates that could be a threat for maintaining genome stability at the final step.

Trinucleotide repeats in the human genome are implicated in various neurodegenerative diseases. The tendency of these repetitive DNA sequences to form non-B DNA structures can cause abnormal replication, leading to genomic instability. This instability contributes to disease progression, though the underlying mechanisms are not fully understood. We investigated the replication of DNA containing CAG and CTG trinucleotide repeats using individual components of the T7 bacteriophage replication machinery, as well as the complete replisome. Our results show that repeats in linear single-stranded DNA (ssDNA) inhibit the activity of T7 DNA polymerase and ssDNA-binding proteins, with a more pronounced effect observed in CTG repeats compared to CAG repeats. Minicircle templates containing CTG repeats exhibited robust DNA synthesis on both the leading and lagging strands, though synthesis was not enhanced by the T7 gene 2.5 ssDNA-binding protein. The lagging strand products generated from the CTG repeat minicircle were significantly longer than those from random sequence templates, and their lengths were not extended by the presence of T7 gene 2.5 protein. When the repeated sequences were incorporated into the T7 phage genome, heterogeneity was observed downstream of the repeats, depending on their length. We propose that aberrant extension occurs predominantly in the lagging strand, driven by dynamic interactions between the repeated sequences and the DNA replisome. This study may provide a foundation for understanding the mechanisms underlying the extension or deletion of repetitive genomic regions.