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.

Mutations mediated by the APOBEC3 (A3) family of single-strand specific cytosine deaminases can accumulate in various cancers, as strand-coordinated clusters and isolated lesions. A3-mediated mutations also occur during normal development, accounting for ~20% of heritable mutations. A3B is an archetypical member of this family and is thought to contribute to both cancer initiation and progression. A3B has a strong preference for C in a TC context and catalyzes hydrolysis of the primary amine of un-paired C to generate U. Subsequent repair generates a distinctive pattern of C-substitutions, which along with their context signify their A3B origin. Although single-stranded DNA is the preferred A3B substrate, we report here that in some instances A3B can deaminate the C of TC in a double-stranded DNA context in vitro. These include C paired to O6-methylguanine (O6meG), to an abasic (AP) site, or to a G adjacent to an AP site. AP sites are the most common lesion in DNA, and O6meG levels increase under alkylating conditions caused by environmental nitrosamines and some chemotherapeutic agents. We also show that elevated expression of A3B can enhance double-stranded breaks induced by the alkylating agent MNNG in mammalian cells, but this effect does not require A3B deaminase activity.

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

Non-enzymatic spontaneous deamination of 5-methylcytosine, producing thymine, is the proposed etiology of cancer mutational signature 1, which is the most predominant signature in all cancers. Here, the proposed mutational process was reconstituted using synthetic DNA and purified proteins. First, single-stranded DNA containing 5-methylcytosine at CpG context was incubated at an elevated temperature to accelerate spontaneous DNA damage. Then, the DNA was treated with uracil DNA glycosylase to remove uracil residues that were formed by deamination of cytosine. The resulting DNA was then used as a template for DNA synthesis by yeast DNA polymerase {delta}. The DNA products were analyzed by next-generation DNA sequencing, and mutation frequencies were quantified. The observed mutations after this process were exclusively C>T mutations at CpG context, which was very similar to signature 1. When 5-methylcytosine modification and uracil DNA glycosylase were both omitted, C>T mutations were produced on C residues in all sequence contexts, but these mutations were diminished by uracil DNA glycosylase-treatment. These results indicate that the CpG>TpG mutations were produced by the deamination of 5-methylcytosine. Additional mutations, mainly C>G, were introduced by yeast DNA polymerase {zeta} on the heat-damaged DNA, indicating that G residues of the templates were also damaged. However, the damage on G residues was not converted to mutations with DNA polymerase {delta} or {varepsilon}. These results provide biochemical evidence to support that the majority of mutations in cancers are produced by ordinary DNA replication on spontaneously damaged DNA.

Microhomology-mediated end joining (MMEJ) is a mutagenic DNA double-strand break (DSB) repair pathway, typically regarded as a backup mechanism in cancer, activated when canonical repair pathways such as non-homologous end joining (c-NHEJ) or homologous recombination (HR) are compromised. While MMEJ has been detected in normal tissues, its presence is puzzling given its error-prone nature, and its physiological role remains poorly defined. Recent studies implicating MMEJ in mitosis suggest a potential function in normal proliferative cells. Here, we show that MMEJ is not uniformly active across tissues but is selectively enriched in proliferative tissues, including thymus, spleen, testes, and liver, while markedly reduced in post-mitotic tissues such as brain, heart, kidney, and lung. This differential activity is supported by tissue-specific expression of key MMEJ components (e.g., Ligase III, MRE11, XRCC1, PARP1, Pol {theta}) and inhibitory factors (e.g., WRN, RAD51, ATM). Moreover, proliferative tissues preferentially utilize short microhomologies ([~]10 nt), whereas post-mitotic tissues rely on longer microhomologies ([≥]13 nt), indicating a shift in repair pathway choice. These findings reveal that MMEJ is a tightly regulated, context-dependent repair pathway. Its activity is tolerated in proliferative tissues due to ongoing cell turnover, while its suppression in long-lived, post-mitotic cells is likely essential to preserve genomic stability. This study assigns a physiological role to MMEJ in healthy tissue homeostasis and highlights its relevance for designing targeted DNA repair-based therapeutic strategies across diverse tissue types.

Repair of DNA damage begins with the elicitation of targeted cellular responses to restore the genome. In E. coli, major products of DNA damage result in the buildup of single-stranded DNA (ssDNA) that is rapidly bound by cooperative filamentation of RecA to initiate the SOS response. The replicative helicase, DnaB, is a central component of the replisome, unwinding duplex DNA in concert with Pol III template dependent synthesis. Interestingly, helicase unwinding is heavily regulated, and the unwinding rate can be reduced by over 10-fold if DnaB becomes decoupled from Pol III. However, if DnaB is dysregulated by mutations that enforce a faster more constricted conformation, unwinding can continue independently, generating excess ssDNA resulting in severe cellular stress. This surplus ssDNA can stimulate RecA recruitment for recombinational repair or activation of SOS to increase the available repair protein pool. To better understand the consequences of dysregulated unwinding, we combined targeted dnaB mutations with an inducible plasmid-based RecA filament inhibition strategy to examine the dependencies on RecA in counteracting decoupling. We find that RecA filamentation is instrumental for processing daughter strand gaps left behind from decoupled unwinding and synthesis to prevent DNA breaks. Without functional RecA filaments, dnaB mutant strains had a greater burden from endogenous damage but without a compensatory increase in mutagenesis. Overall, RecA plays a critical role in strain survival by processing DNA gaps and protecting from breaks caused by dysregulated or interrupted helicase activity in vivo. AUTHOR SUMMARYCoupled DNA unwinding and synthesis is a genomic protection strategy used during DNA replication to prevent excessive buildup of labile single-stranded DNA (ssDNA). The helicase and polymerase enzymes have evolved multidimensional regulation tactics to maintain this connection despite different individual kinetic rates and differential responses to genomic obstacles. For one, the DnaB helicase in E. coli can alter its hexameric ring structure by dilating to slow down or constricting to speed up DNA unwinding. Here, we have utilized persistently constricted mutants of DnaB in vitro or genomically edited dnaB in vivo to induce decoupling in the replisome. Constricted DnaB mutants limit total leading strand synthesis by Pol III, indicating that lost kinetic regulation between these enzymes results in inefficient replication. Using an inducible plasmid-based system to disrupt Rad51 filamentation in vivo, we show that RecA is responsible for the increased mutagenesis, a filamented cellular phenotype, and mitigating DNA breaks from excess ssDNA caused by decoupling. These results reveal a role for RecA filamentation in mediating excess ssDNA resulting from decoupling to maintain survival and adaptation.

In the event of DNA damage, the cell cycle can be slowed or halted to allow for DNA repair. The mechanisms by which this occurs are well-characterised in interphase, although the mechanisms underpinning mitosis slowing in response to damage are unclear. Canonical checkpoints and DNA repair pathways are largely repressed in mitosis, and whilst there is some level of mitotic DNA synthesis and repair, the bulk of DNA damage is processed for post-mitotic repair. How the decision is made between mitotic DNA repair and post-mitotic DNA repair is not known. We have identified the antioxidant enzyme Superoxide Dismutase 1 (SOD1) as an essential factor mediating delayed mitotic progression in response to DNA damage and replication stress. Cells depleted of SOD1 no longer exhibit DNA damage dependent mitotic delay, and display increased levels of damaged centromeres and mitotic defects. Whilst reactive oxygen species (ROS)-inducing agents also lead to SOD1-dependent mitotic delay, intracellular ROS levels do not correlate with mitotic arrest. SOD1 appears to play an important role in DNA repair in interphase and is recruited to the nucleus in response to DNA damage. In addition to control of mitotic progression in response to genotoxic stress, SOD1 also plays a major role in mitotic DNA synthesis. SOD- depleted cells show reduced levels of mitotic EdU incorporation in response to either replication stress or DNA breaks, seemingly in tandem with Rad51 andSOD1-depletion induced mitotic progression in the presence of DNA breaks is Rad52-dependent. We suggest that there are two responses to DNA breaks in mitosis; either arrest and mitotic repair or progression and post-mitotic repair; and these two pathways exist in a fine balance, controlled by a signaling cascade involving SOD1.

Deinococcus radiodurans, a natural transformation (NT) enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavate into the intricate roles of NT machinery in the stressed D. radiodurans, focusing on the genes comEA, comEC, endA, pilT and dprA, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveals that NT not only supports the nutritional needs of D. radiodurans under stress but also have roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and EndA) might contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells while DprA contribute differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be one of factor in D. radiodurans remarkable ability to survive and thrive in environments characterized by high levels of DNA-damaging agents. Author SummaryDeinococcus radiodurans, a bacterium known for its extraordinary radiation resistance. This study explores the roles of natural transformation (NT) machinery in the radiation-resistant bacterium Deinococcus radiodurans, focusing on the genes comEA, comEC, endA, pilT, and dprA. These genes are crucial for the uptake and processing of extracellular DNA (eDNA) and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhance D. radiodurans survival in environments with high levels of DNA-damaging agents.

Praja1 is known as an E3 ubiquitin ligase that regulates multiple functions through protein degradation. It acquired nuclear localization signal after gene duplication and although studies have shown some significant roles of nuclear Praja1, comprehensive analysis still lacks. In this study, we performed comparative proteomics and biochemical analyses to elucidate the functions of Praja1 in the nucleus. First, proteomics analysis applied to nuclear localization deficient Praja1 exhibited signs of DNA damage response fluctuation. Subsequent comet assay revealed Praja1 protecting cells from various DNA damage sources. Similarly, cells lacking Praja1 became more sensitive to DNA damage-induced cell death, while E. coli expressing Praja1 exhibited resistance to DNA damage. To further elucidate the molecular basis of DNA protection, gel shift assay showed direct binding of Praja1 to DNA through electrostatic interactions within its intrinsically disordered region. Further in vitro damaging assay suggested that Praja1 may induce structural changes that enhance DNA repair efficiency upon binding to DNA. Together, these results provide insights into the evolutionarily novel role of nuclear Praja1 in protecting against DNA damage.

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged DNA fragments. In this study, we investigate the regulatory role of the NT-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks (DSBs), with diminished involvement of the ESDSA pathway. Furthermore, our data suggest that the SSA pathway becomes the primary mechanism for DNA DSB repair in the absence of DprA. Overall, our results highlight the regulatory function of DprA in modulating the choice between SSA and ESDSA pathways for DNA repair in the radiation-resistant bacterium D. radioduransx.

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

G-quadruplex (G4) structures are non-B DNA elements enriched within the mitochondrial genome and serve as substrates for Endonuclease G (EndoG). Under oxidative stress, endo G relocates from the intermembrane space to the matrix, where it cleaves G4 motifs and generates mitochondrial double-strand breaks (DSBs). Notably, mitochondrial DNA (mtDNA) frequently harbours large deletions flanked by G4 motifs; these deletions are widespread in ageing and mitochondrial disorders, yet the mechanistic basis of their formation remains poorly understood. Here, we identify a damage-specific and stress-responsive mtDNA repair program that resolves EndoG-induced DSBs, using biochemical reconstitution and pharmacological inhibition. We show that these breaks are primarily repaired via microhomology-mediated end joining (MMEJ) and homologous recombination (HR), facilitated by the mitochondrial recruitment of canonical factors, including PARP1, MRE11, and Ligase III. Inhibition of PARP1 or MRE11 significantly impairs repair efficiency, confirming their essential roles in mitochondrial DSB resolution. Interestingly, exposure to ionising radiation (5 Gy) selectively suppresses mitochondrial MMEJ while enhancing HR, revealing a compensatory pathway switch tuned to the nature and severity of genotoxic stress. Classical nonhomologous ending (cNHEJ) remains undetectable under all conditions. Collectively, our findings delineate a flexible, lesion-dependent mitochondrial repair network that resolves DSBs via error-prone or recombinogenic mechanisms. This work provides mechanistic insight into the origin of mtDNA deletions and highlights the adaptive plasticity of mitochondrial genome maintenance under physiological and genotoxic stress.

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.

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.

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.

Various DNA repair machineries have evolved in the cell to maintain the integrity of the genome for proper functioning of the same. Repair of ultraviolet (UV) irradiation mediated DNA damage occurs by nucleotide excision repair pathway through transcription coupled repair (TCR), a process in which the damage is repaired on transcriptionally active genic regions. This process requires various protein complexes including heterodimer DNA Damage Binding 1 (DDB1) protein. Defects in TCR have been found in patients with mutations in the Cockayne syndrome (CS) group A and group B genes and in the Xeroderma pigmentosum (XP) group G gene. Transcription factors (TFs) play a very important role in regulation of TCR system, especially those TFs which binds to DNA at specific loci. Several TFs have been shown to modulate the repair of photolesions either by inducing or inhibiting the TCR. However, the mechanism behind their action is not very clear. Mammalian TTF1 is an essential multifunctional transcription factor involved in transcription initiation, termination, DNA fork blockage, chromatin remodelling etc., and has been shown to interact with CSB protein. Hence, to discover its role in TCR and to identify its interaction partners, we purified this protein and did a pull-down assay with HEK293T cell lysate followed by LC-MS and discovered DDB1 as one of its major interactors. This established our confidence that TTF1 protein might be playing a critical role in TCR. Further, we discovered that, upon UV mediated DNA damage in HEK293T cells the expression of TTF1 is significantly induced and is co-localized with {gamma}H2AX protein. To our surprise, we found that after knockdown of TTF1, DDB1 level decreases in HEK293T cells while knockdown of DDB1, increases TTF1 level in the cells. Hence, our study opens up a new avenue towards exploring a noble function of the transcription factor TTF1, which in turn could establish the potential to develop therapeutics towards cancers and other diseases.

Current models for Okazaki fragment maturation in eubacteria invoke RNA cleavage by RNase H, followed by strand displacement synthesis and 5' RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 5'-3' flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on 5' flapped and nicked RNA-DNA hybrid substrates. We found that the 5' nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 5' flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that {Delta}polA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 5' nuclease domain fails to complement {Delta}polA. Cells lacking FEN ({Delta}fenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model is similar to models for Okazaki fragment processing in eukaryotes, where Pol d catalyzes strand displacement synthesis followed by 5' flap cleavage using FEN-1. Author Summary5' flap endo/exonuclease (FEN) activity provides an essential contribution to DNA replication and repair in all cellular life. In bacteria, DNA polymerase I is thought to be the central enzyme involved in Okazaki fragment processing, using its DNA polymerase and 5' nuclease activities to generate and then remove the 5 ssRNA segment of an Okazaki fragment. Many bacterial genomes encode a second, discrete FEN in addition to Pol I. We show that FEN is the primary 5' nuclease used by B. subtilis for primer removal. FEN activity exceeds that of Pol I on most substrates, including several that mimic Okazaki fragment intermediates. Additionally, we provide genetic evidence showing that FEN is involved in Okazaki fragment processing and that it is the DNA polymerase domain of Pol I rather than its 5' nuclease domain that is important in vivo. With our results, we propose a new model for Okazaki fragment processing in B. subtilis, which may be prevalent in a wider group of bacteria.

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.

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.

Oxidative stress, generated by both endogenous and exogenous agents, can cause DNA lesions that, if not repaired, accumulate as somatic mutations and can contribute to cancer initiation. Here, we explored this problem through the lens of DNA electronic properties, quantified by the vertical ionization potential (vIP) of nucleobase motifs, which reflects their susceptibility to oxidation. We analyzed genome-wide experimental data on oxidative DNA damage and found that the highest damage levels occur in regions with low vIP values, supporting a causal link between them. The analysis of cancer mutational signatures and their annotated aetiologies revealed strong anticorrelations between mutation frequency and vIP values, particularly in cancers driven by oxidative DNA damage, such as lung cancer. We further computed anticorrelations between vIP values and the frequencies of mutated motifs across coding and non-coding regions and across different mutation types, observing the strongest anticorrelations for silent mutations, consistent with their reduced selective pressure. Moreover, similar anticorrelations were observed for somatic mutations in cancer and normal tissues, as well as for germline mutations, suggesting that they arise from similar mutagenesis processes. This work clarifies how oxidative damage, DNA electronic properties and carcinogenesis are related and help identify genomic regions more prone to mutations.