Hydrogen peroxide is a precursor to reactive oxygen species (ROS) in cells because of its high reactivity with iron(II) carbonate complexes formed in the labile iron pool due to a high concentration of intracellular bicarbonate (25-100 mM). This chemistry leads to the formation of carbonate radical anion rather than hydroxyl radical, and unlike the latter ROS, CO is a milder one-electron oxidant with high specificity for guanine oxidation in DNA and RNA. In addition to metabolism, another major source of DNA oxidation is inflammation which generates peroxynitrite, another precursor to CO via reaction with dissolved CO. The identity of the ROS is important because not all radicals react with DNA in the same way. Whereas hydroxyl radical forms adducts at all four bases and reacts with multiple positions on ribose leading to base loss and strand breaks, carbonate radical anion is focused on guanosine oxidation to yield 8-oxo-7,8-dihydroguanosine in nucleic acids and the nucleotide pool, a modification that can function epigenetically in the context of a G-quadruplex. DNA sequences of multiple adjacent guanines, as found in G-quadruplex-forming sequences of gene promoters, are particularly susceptible to oxidative damage, and the focusing of CO chemistry on these sites can lead to a transcriptional response during base excision repair. In this pathway, AP-endonuclease 1 plays a key role in accelerating G-quadruplex folding as well as recruiting activating transcription factors to impact gene expression.

In recent years, numerous reports indicated that, besides pathogen infections, DNA replication stress and defective DNA repair can trigger the innate immune response by introducing a state of viral mimicry, due to cytosolic accumulation of the self-nucleic acid species, which culminates in the activation of type I interferon (IFN) pathway. In turn, IFN upregulates a variety of factors mutually implicated in immune- and genome-related mechanisms, shedding light on the unprecedented causality between genome stability and innate immunity. Intriguingly, in addition to being induced by replication stress, IFN-regulated factors can also promote it, pinpointing IFN signaling as both a consequence and a cause of replication stress. Here, we provide an overview of the factors and molecular mechanisms implicated in the evolutionary conserved crosstalk between genome maintenance and innate immunity, highlighting the role of the IFN-stimulated gene 15 (ISG15), which appears to be at the hub of this intersection. Moreover, we discuss the potential significance and clinical implications of the immune-mediated modulation of DNA replication and repair upon pathogen infection and in human diseases such as cancer and autoinflammatory syndromes. Finally, we discuss the relevant open questions and future directions.

Rad51 filaments are Rad51-coated single-stranded DNA and essential in homologous recombination (HR). The yeast Shu complex (Shu) is a conserved regulator of homologous recombination, working through its modulation on Rad51 filaments to direct HR-associated DNA damage response. However, the biochemical properties of Shu remain unclear, which hinders molecular insights into Shu's role in HR and the DNA damage response. In this work, we biochemically characterized Shu and analyzed its molecular actions on single-stranded DNA and Rad51 filaments. First, we revealed that Shu preferentially binds fork-shaped DNA with 20nt ssDNA components. Then, we identified and validated, through site-specific mutagenesis, that Shu is an ATPase and hydrolyzes ATP in a DNA-dependent manner. Furthermore, we showed that Shu interacts with ssDNA and Rad51 filaments and alters the properties of ssDNA and the filaments with a 5'-3' polarity. The alterations depend on the ATP hydrolysis of Shu, suggesting that the ATPase activity of Shu is important in regulating its functions. The preference of Shu for acting on the 5' end of Rad51 filaments aligns with the observation that Shu promotes lesion bypass at the lagging strand of a replication fork. Our work on Shu, a prototype modulator of Rad51 filaments in eukaryotes, provides a general molecular mechanism for Rad51-mediated error-free DNA lesion bypass.

Repeat-mediated deletions (RMDs) are a type of deletion rearrangement that utilizes two repetitive elements to bridge a DNA double-strand break (DSB) that leads to loss of the intervening sequence and one of the repeats. Sequence divergence between repeats causes RMD suppression and indeed this divergence must be resolved in the RMD products. The mismatch repair factor, MLH1, was shown to be critical for both RMD suppression and a polarity of sequence divergence resolution in RMDs. Here, we sought to study the interrelationship between these two aspects of RMD regulation (i.e., RMD suppression and polar divergence resolution), by examining several mutants of MLH1 and its binding partner PMS2. To begin with, we show that PMS2 is also critical for both RMD suppression and polar resolution of sequence divergence in RMD products. Then, with six mutants of the MLH1-PMS2 heterodimer, we found several different patterns: three mutants showed defects in both functions, one mutant showed loss of RMD suppression but not polar divergence resolution, whereas another mutant showed the opposite, and finally one mutant showed loss of RMD suppression but had a complex effect on polar divergence resolution. These findings indicate that RMD suppression vs. polar resolution of sequence divergence are distinct functions of MLH1-PMS2.

Ubiquitin-specific protease 1 (USP1) is the founding member of the family of cysteine proteases that catalyse hydrolysis of the isopeptide bond between ubiquitin and targets. USP1 is often overexpressed in various cancers, and expression levels correlate with poor prognosis. USP1 and its partner USP1-associated Factor 1 (UAF1) are required for deubiquitinating monoubiquitin signals in DNA interstrand crosslink repair, and in Translesion synthesis, among others, and both proteins are subject to multiple regulations themselves. This review covers recent findings on the mechanisms and functions of USP1 in DNA repair, its regulation, and its potential as a target for therapeutic intervention.

Tandem duplications (TD) are among the most frequent type of structural variant (SV) in the cancer genome. They are characterized by a single breakpoint junction that defines the boundaries and the size of the duplicated segment. Cancer-associated TDs often increase oncogene copy number or disrupt tumor suppressor gene function, and thus have important roles in tumor evolution. TDs in cancer genomes fall into three classes, defined by the size of duplications, and are associated with distinct genetic drivers. In this review, we survey key features of cancer-related TDs and consider possible underlying mechanisms in relation to stressed DNA replication and the 3D organization of the S phase genome.

The MSH7 protein is a binding partner of MSH2 forming the MutSγ complex. This complex contributes to the plant mismatch repair (MMR) system by recognizing DNA base-base mismatches. Here, we evaluated the impact of MSH7 on genetic diversity of the tenth generation (G) of wild type and MSH7 deficient Arabidopsis thaliana plants before and after two days exposure to 100 mM NaCl. Genetic diversity was assessed using inter simple sequence repeats (ISSR) and high-resolution melting (HRM) analyses. ISSR analyses revealed a 6.7 % or 5.8 % average polymorphism in the G of wild type before and after a short-term salt stress, respectively, and a 64.4 % or 72.1 % average polymorphism in the G of msh7 mutant plants before and after salt treatment, respectively. Interestingly, several ISSR markers showed different polymorphism patterns after salt stress compared with the control before treatment. We next compared the percentage of the G of wild type and msh7 seedlings with polymorphic bands. Statistically significant differences between genotypes but not due to the salt treatment were observed. In addition, co-amplification at lower temperature-PCR followed by HRM analysis was performed. Of the five assayed HRM loci, two loci allowed the discrimination of fragment alleles between genotypes and two loci, between conditions. We conclude that MSH7 deficient A. thaliana mutants accumulated mutations over 10 generations, and that two days of salt stress caused a further increase in new mutations, thus enhancing genetic diversity that may favor new traits associated with stress tolerance, fitness, and adaptation.

Most giant viruses including Mimiviridae family build large viral factories within the host cytoplasms. These giant viruses are presumed to possess specific genes that enable the rapid and massive replication of their large double-stranded DNA genomes within viral factories. It has been revealed that a functionally uncharacterized protein, MutS7, is expressed during the operational phase of the viral factory. MutS7 contains an N-terminal mismatched DNA-binding domain, which is similar to the mismatched DNA-recognizing protein MutS1, and a unique C-terminal HNH endonuclease domain absent in other MutS family proteins. MutS7 gene of the genus Mimivirus of the family Mimiviridae is encoded in the locus that is responsible for resistance against infection of a virophage. In the present study, we characterized the MutS7 HNH domain of Mimivirus shirakomae. The HNH domain preferentially bound to branched DNA structures containing single-stranded regions, especially the displacement-loop structure, which is a primary intermediate in homologous/homeologous recombination, rather than to linear DNAs and branched DNAs lacking single-stranded regions. However, the HNH domain exhibited no endonuclease activity. The site-directed mutagenesis analysis revealed that the Cys4-type zinc finger of the HNH domain was not essential, but was important for the DNA binding. Given that giant virus MutS7 contains a mismatch-binding domain in addition to the HNH domain, we propose that giant virus MutS7 may suppress homeologous recombination in the viral factory.

Quantitative genomic mapping of DNA damage may provide insights into the underlying mechanisms of damage and repair. Sequencing based approaches are bound to the limitations of PCR amplification bias and read length which hamper both the accurate quantitation of damage events and the ability to map them to structurally complex genomic regions. Optical Genome mapping in arrays of parallel nanochannels allows physical extension and genetic profiling of millions of long genomic DNA fragments, and has matured to clinical utility for characterization of complex structural aberrations in cancer genomes. Here we present a new mapping modality, Repair-Assisted Damage Detection - Optical Genome Mapping (RADD-OGM), a method for single-molecule level mapping of DNA damage on a genome-wide scale. Leveraging ultra-long reads to assemble the complex structure of a sarcoma cell-line genome, we mapped the genomic distribution of oxidative DNA damage, identifying regions more susceptible to DNA oxidation. We also investigated DNA repair by allowing cells to repair chemically induced DNA damage, pinpointing locations of concentrated repair activity, and highlighting variations in repair efficiency. Our results showcase the potential of the method for toxicogenomic studies, mapping the effect of DNA damaging agents such as drugs and radiation, as well as following specific DNA repair pathways by selective induction of DNA damage. The facile integration with optical genome mapping enables performing such analyses even in highly rearranged genomes such as those common in many cancers, a challenging task for sequencing-based approaches.

Mitochondrial DNA (mtDNA) is often more susceptible to damage compared to nuclear DNA. This is due to its localization in the mitochondrial matrix, where a large portion of reactive oxygen species are produced. Mitochondria do not have histones and mtDNA is only slightly protected by histone-like proteins and is believed to have less efficient repair mechanisms. In this review, we discuss the long-range PCR method, which allows for the effective detection of mtDNA damage. The method is based on the assumption that various types of DNA lesions can interfere the progress of DNA polymerase, resulting in reduced amplification efficiency. It can be used to estimate the number of additional (above background) lesions in mtDNA. The review outlines the evolution of the methodology, its variations, applications in a wide range of model organisms, the advantages of the method and its limitations, as well as ways to overcome these limitations. Over the past two decades, the use of long-range PCR has allowed the study of mtDNA repair mechanisms, the characteristics of mitochondrial genome damage in various neurodegenerative diseases, aging, ischemic and oncological processes, as well as in anticancer therapy. The assessment of mtDNA damage has also been proposed for use in environmental biomonitoring. This review provides a critical evaluation of the various variations of this method, summarizes the accumulated data, and discusses the role of mtDNA damage in different organs at the organismal level.

A nucleoside analog, Cidofovir (CDV), is used for the treatment of viral diseases such as cytomegalovirus retinitis and herpes virus infection. CDV converts to its active diphosphate metabolite (CDVpp) through cellular kinases and acts as a competitive inhibitor for viral polymerase thereby interfering with viral replication. However, the effect of this drug on the replication of healthy host cells and the mechanisms involved in the cellular tolerance to CDV are yet to be fully understood. In this study, we explored the mechanisms underlying cellular tolerance to CDV by screening mutant cell lines exhibiting hypersensitivity to CDV from a collection of DT40 mutants deficient in various genome maintenance systems. We identified Rad17 and PrimPol as critical factors for CDV tolerance. We found that Rad17 plays a pivotal role in activating intra-S phase checkpoint by the phosphorylation of Chk1, a vital checkpoint mediator. We showed that PrimPol, a factor involved in the release of stalled replication, plays critical roles in CDV tolerance in tandem with Rad17. We found that PrimPol deficient cells showed slower replication on the CDV-incorporated template strand than did wild-type cells, indicating a critical role of PrimPol in the continuous replication fork progression on the CDV-incorporated damaged template. PrimPol releases replication arrest with its DNA-damage bypass function and its repriming function, we thus investigated which PrimPol function is involved in CDV tolerance using the separation of function mutant genes of PRIMPOL. The CDV hypersensitive phenotype of PrimPol deficient cells was restored by PRIMPOL (primase active / reduced polymerase activity), indicating that the repriming function of PrimPol is required for maintaining replication on the CDV-damaged template. Moreover, we found that the number of sister chromatid exchange (SCE) was reduced in PrimPol-deficient cells. These data indicate that gaps generated by PrimPol-mediated repriming on CDV-damaged templates promote post-replicative gap-filing by template switching.

DNA double-strand breaks (DSBs) trigger the recruitment of repair protein and promote signal transduction through posttranslational modifications such as phosphorylation. After DSB induction, ataxia telangiectasia mutated (ATM) phosphorylates H2AX on chromatin surrounds the mega-base pairs proximal to the DSBs. Advanced super-resolution microscopic technology has demonstrated the formation of γH2AX nano foci as a unit of nano domain comprised of multiple nucleosomes. The formation of γH2AX nano foci could be potentially affected by pre-existing chromatin structure prior to DSB induction; however, it remains unclear whether chromatin status around DSBs influences the formation of γH2AX nano foci. In this study, to investigate γH2AX nano foci formation in the context of chromatin relaxation, γH2AX nano foci were examined following the depletion of MeCP2, which is a factor promoting chromatin condensation. Remarkably, by using super-resolution imaging analysis, we found that the volume of γH2AX nano foci cluster in MeCP2-depleted cells was significantly greater than that in control cells, both 5 and 30 min after ionizing radiation (IR). Corresponding to the increased volume size, the number of γH2AX nano foci per cluster was greater than that in control cells, while the distance of each nano focus within foci clusters remained unchanged. These findings suggest that relaxed chromatin condition by MeCP2 depletion facilitates faster and more extensive γH2AX nano foci formation after IR. Collectively, our super-resolution analysis suggests that the chromatin status surrounding DSBs influences the expansion of γH2AX nano foci formation, thus, potentially influencing the DSB repair and signaling.

Proper chromatin remodeling is crucial for many cellular physiological processes, including the repair of DNA double-strand break (DSB). While the mechanism of DSB repair is well understood, the connection between chromatin remodeling and DSB repair remains incompletely elucidated. In this review, we aim to highlight recent studies demonstrating the close relationship between chromatin remodeling and DSB repair. We summarize the impact of DSB repair on chromatin, including nucleosome arrangement, chromatin organization, and dynamics, and conversely, the role of chromatin architecture in regulating DSB repair. Additionally, we also summarize the contribution of chromatin remodeling complexes to cancer biology through DNA repair and discuss their potential as therapeutic targets for cancer.

DNA double-strand breaks (DSBs) are cytotoxic lesions that compromise genomic integrity and trigger cell death. Homologous recombination (HR) is a major pathway for repairing DSBs in cycling cells. However, it remains unclear whether transient modulation of HR could confer protection to adult stem cells against lethal irradiation exposure. In this study, we investigated the radio-protective effect of the RAD51-stimulatory compound RS-1 on adult stem cells and progenitor cells with varying cycling rates in intestinal and hematopoietic tissues. Treatment with RS-1 even at high doses did not induce noticeable cell death or proliferation of intestinal crypt cells in vivo. Pretreatment with RS-1 before irradiation significantly decreased mitotic death, promoted DNA repair and enhanced the survival of intestinal stem cells and progenitor cells and increased the number of regenerative crypt colonies thereby mitigating IR-induced gastrointestinal syndrome. Moreover, RS-1 pretreatment could increase the survival and regeneration of irradiated intestinal organoids in vitro, which can be rescued by RAD51 inhibitor. However, pretreatment with RS-1 in vivo did not elevate nucleated cell count or HSPCs in bone marrow after 6 Gy irradiation. Additionally, there was no impact on mouse survival due to drug treatment observed. Thus, our data suggest that targeting HR as a strategy to prevent tissue damage from acute irradiation exposure may depend on cell cycling rates and intrinsic DNA repair mechanisms.

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

The Repeat Expansion Diseases (REDs) are a large group of human genetic disorders that result from an increase in the number of repeats in a disease-specific tandem repeat or microsatellite. Emerging evidence suggests that the repeats trigger an error-prone form of DNA repair that causes the expansion mutation by exploiting a limitation in normal mismatch repair. Furthermore, while much remains to be understood about how the mutation causes pathology in different diseases in this group, there is evidence to suggest that some of the downstream consequences of repeat expansion trigger the DNA damage response in ways that contribute to disease pathology. This review will discuss these subjects in the context of the Fragile X-related disorders (aka the FMR1 disorders) that provide a particularly interesting example of the intersection between the repeats and the DNA damage response that may also be relevant for many other diseases in this group.

Acetaldehyde (AcH) is the first metabolite of ethanol and is proposed to be responsible for the genotoxic effects of alcohol consumption. As an electrophilic aldehyde, AcH can form multiple adducts with DNA and other biomolecules, leading to function-altering and potentially toxic and carcinogenic effects. In this review, we describe sources of AcH in humans, including AcH biosynthesis mechanisms, and outline the structures, properties and functions of AcH-derived adducts with biomolecules. We also describe human AcH detoxification mechanisms and discuss ongoing challenges in the field.