Search

The DNA damage response (DDR) ensures error-free DNA replication and transcription and is disrupted in numerous diseases. An ongoing challenge is to determine the proteins orchestrating DDR and their organization into complexes, including constitutive interactions and those responding to genomic insult. Here, we use multi-conditional network analysis to systematically map DDR assemblies at multiple scales. Affinity purifications of 21 DDR proteins, with/without genotoxin exposure, are combined with multi-omics data to reveal a hierarchical organization of 605 proteins into 109 assemblies. The map captures canonical repair mechanisms and proposes new DDR-associated proteins extending to stress, transport, and chromatin functions. We find that protein assemblies closely align with genetic dependencies in processing specific genotoxins and that proteins in multiple assemblies typically act in multiple genotoxin responses. Follow-up by DDR functional readouts newly implicates 12 assembly members in double-strand-break repair. The DNA damage response assemblies map is available for interactive visualization and query (ccmi.org/ddram/).

Simple Summary: Non-Hispanic Black breast cancer survivors have poorer outcomes than White survivors, but the biological mechanisms underlying these disparities are unclear. We discovered novel race-based differences in systemic DNA damage and repair activity among breast cancer survivors. This finding suggests DNA damage and repair are important basic science mechanisms in cancer disparities. Non-Hispanic Black breast cancer survivors have poorer outcomes and higher mortality rates than White survivors, but systemic biological mechanisms underlying these disparities are unclear. We used circulating leukocytes as a surrogate for measuring systemic mechanisms, which might be different from processes in the target tissue (e.g., breast). We investigated race-based differences in DNA damage and repair, using a novel CometChip assay, in circulating leukocytes from breast cancer survivors who had completed primary cancer therapy and were cancer free. We observed novel race-based differences in systemic DNA damage and repair activity in cancer survivors, but not in cells from healthy volunteers. Basal DNA damage in leukocytes was higher in White survivors, but Black survivors showed a much higher induction after bleomycin treatment. Double-strand break repair activity was also significantly different between the races, with cells from White survivors showing more sustained repair activity compared to Black leukocytes. These results suggest that cancer and cancer therapy might have long-lasting effects on systemic DNA damage and repair mechanisms that differ in White survivors and Black survivors. Findings from our preliminary study in non-cancer cells (circulating leukocytes) suggest systemic effects beyond the target site, with implications for accelerated aging-related cancer survivorship disparities. [ABSTRACT FROM AUTHOR]

Background: Microcephaly with early‐onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3′‐phosphatase (PNKP). Methods: We have used whole genome sequencing and Sanger sequencing to identify disease‐causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP‐ribose immunofluorescence. Results: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single‐strand break repair, confirming that the mutated PNKP alleles are dysfunctional. Conclusion: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1‐induced single‐strand breaks, and most likely causes MCSZ in this patient. [ABSTRACT FROM AUTHOR]

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types. [ABSTRACT FROM AUTHOR]

DNA single-strand breaks (SSBs) are amongst the commonest DNA lesions arising in cells, with many tens of thousands induced in each cell each day. SSBs arise not only from exposure to intracellular and environmental genotoxins but also as intermediates of normal DNA metabolic processes, such as the removal of torsional stress in DNA by topoisomerase enzymes and the epigenetic regulation of gene expression by DNA base excision repair (BER). If not rapidly detected and repaired, SSBs can result in RNA polymerase stalling, DNA replication fork collapse, and hyperactivation of the SSB sensor protein poly(ADP-ribose) polymerase 1 (PARP1). The potential impact of unrepaired SSBs is illustrated by the existence of genetic diseases in which proteins involved in SSB repair (SSBR) are mutated, and which are typified by hereditary neurodevelopmental and/or neurodegenerative disease. Here, I review our current understanding of SSBR and its impact on human neurological disease, with a focus on recent developments and concepts. DNA single-strand breaks (SSBs) are very common lesions, and mutations in SSB repair (SSBR) genes result in multiple genetic neurological diseases typified by neurodevelopmental dysfunction and/or neurodegeneration. SSBR comprises several interlinked pathways that detect and process SSBs of a broad range of sources and chemistries, resulting from stochastic DNA damage and from intrinsic errors during 'programmed' DNA metabolic processes such as DNA replication, transcription, and epigenetic reprogramming. SSBs are detected by poly(ADP-ribose) polymerase 1 (PARP1) and PARP2, which regulate chromatin remodelling, transcription, and the recruitment of SSBR factors such as XRCC1 protein complexes during SSBR. If SSBs accumulate or persist excessively, hyperactivation of the SSB sensor PARP1 can lead to excessive NAD+ depletion, to poly(ADP-ribosylation), and to neurological dysfunction. [ABSTRACT FROM AUTHOR]

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

UV irradiation induces "bulky" DNA photodimers such as (6-4)-photoproducts and cyclobutane pyrimidine dimers that are removed by nucleotide excision repair, a complex process defective in the sunlight-sensitive and cancer-prone disease xeroderma pigmentosum. Some bacteria and lower eukaryotes can also repair photodimers by enzymatically simpler mechanisms, but such pathways have not been reported in normal human cells. Here, we have identified such a mechanism. We show that normal human cells can employ a DNA base excision repair process involving NTH1, APE1, PARP1, XRCC1, and FEN1 to rapidly remove a subset of photodimers at early times following UVC irradiation. Loss of these proteins slows the early rate of repair of photodimers in normal cells, ablates their residual repair in xeroderma pigmentosum cells, and increases UVC sensitivity ∼2-fold. These data reveal that human cells can excise photodimers using a long-patch base excision repair process that functions additively but independently of nucleotide excision repair. [Display omitted] • Human cells employ a BER-dependent pathway to repair a subset of UV-induced photodimers • The repair is triggered by NTH1 and APE1 and requires PARP1, XRCC1, and FEN1 • This alternative pathway functions additively but independently of NER • Loss of this pathway increases the UV sensitivity of human cells ∼2-fold Gautam et al. show for the first time the existence of an alternative pathway to repair UV-induced DNA damage (photodimers) in normal human cells. This pathway functions additively but independently of NER and is human equivalent of the evolutionary-ancient base excision repair-dependent mechanism reported in bacteriophage and some bacteria. [ABSTRACT FROM AUTHOR]

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/ SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (At RAD1), single-strand break repair (At PARP1), as well as microhomology-mediated double-strand break repair (At TEB). Moreover, RAD5A can partially complement for a deficient At ATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3′-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair. [ABSTRACT FROM AUTHOR]

Microscopy and micro-irradiation imaging techniques have significantly advanced our knowledge of DNA damage tolerance and the assembly of DNA repair proteins at the sites of damage. While these tools have been extensively applied to the study of nucleotide excision repair and double-strand break repair, their application to the repair of oxidatively-induced base lesions and single-strand breaks is just beginning to yield new insights. This review will focus on examining micro-irradiation techniques reported to create base lesions and single-strand breaks; these lesions are considered to be primarily addressed by proteins involved in the base excision repair (BER) pathway. By examining conditions for generating these DNA lesions and reviewing information on the assembly and dissociation of repair complexes at the induced lesion sites, we hope to promote further investigations into BER and to stimulate further development and enhancement of these techniques for the study of BER. [ABSTRACT FROM AUTHOR]

Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined. [ABSTRACT FROM AUTHOR]

Abstract: DNA strand breaks arise continuously in cells and can lead to chromosome rearrangements and genome instability or cell death. The commonest DNA breaks are DNA single-strand breaks, which arise at a frequency of tens-of-thousands per cell each day and which can block the progression of RNA/DNA polymerases and disrupt gene transcription and genome duplication. If not rapidly repaired, SSBs can be converted into DNA double-strand breaks (DSBs) during genome duplication, eliciting a complex series of DNA damage responses that attempt to protect cells from irreversible replication fork collapse. DSBs are the most cytotoxic and clastogenic type of DNA breaks, and can also arise independently of DNA replication, albeit at a frequency several orders of magnitude lower than SSBs. Here, I discuss the evidence that DNA single- and double -strand break repair pathways, and cellular tolerance mechanisms for protecting replication forks during genome duplication, utilize signalling by protein ADP-ribosyltransferases to protect cells from the harmful impact of DNA strand breakage. [Copyright &y& Elsevier]

Highlights: [•] Tissue specificity of DNA repair is not well understood and is understudied. [•] TNR instability is ideal to study DNA repair in different tissues. [•] Chromatin, repair rates, and protein levels may account for this tissue specificity. [•] System-level experiments will improve understanding of tissue-specific DNA repair. [Copyright &y& Elsevier]

Abstract: Oxidative DNA damage results from the attack by reactive oxygen and nitrogen species (ROS/RNS) on human genome. This includes base modifications such as oxidized bases, abasic (AP) sites, and single-strand breaks (SSBs), all of which are repaired by the base excision repair (BER) pathway, one among the six known repair pathways. BER-pathway in mammalian cells involves several evolutionarily conserved proteins and is also linked to genome replication and transcription. The BER-pathway enzymes, namely, DNA glycosylases (DGs) and the end-processing proteins such as abasic endonuclease (APE1), form complexes with downstream repair enzymes via protein–protein and DNA–protein interactions. An emerging concept for BER proteins is their involvement in non-canonical functions associated to RNA metabolism, which is opening new interesting perspectives. Various mechanisms that are underlined in maintaining neuronal cell genome integrity are identified, but are inconclusive in providing protection against oxidative damage in neurodegenerative disorders, main emphasis is given towards the role played by the proteins of BER-pathway that is discussed. In addition, mechanisms of action of BER-pathway in nuclear vs. mitochondria as well as the non-canonical functions are discussed in connection to human neurodegenerative diseases. [Copyright &y& Elsevier]

Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104 dizygotic). We did not detect genetic effects on the DNA-strand break variables in our study. Environ. Mol. Mutagen. 54:414-420, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases. Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age-associated changes in DNA damage response and the capacities of repairing DNA single-strand breaks ( SSBs) and double-strand breaks ( DSBs) in human peripheral blood mononuclear cells ( PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity in a study population consisting of 216 individuals from a population-based sample of twins aged 40-77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ-H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach statistical significance after adjustment for batch effect across multiple experiments. No gender differences were observed for any of the parameters analyzed. Our findings suggest that in PBMCs, the repair of SSBs is maintained until old age, whereas the response to and the repair of DSBs decrease. [ABSTRACT FROM AUTHOR]

Abstract: Inbuilt mechanisms of DNA surveillance and repair are integral to the maintenance of genomic stability. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that plays a critical role in DNA damage response processes. PARP inhibition has been successfully employed as a novel therapeutic strategy to enhance the cytotoxic effects of DNA-damaging agents. We have shown that PARP inhibition has substantial single agent antitumour activity with a wide therapeutic index in homologous DNA repair-defective tumours such as those arising in BRCA1 and BRCA2 mutation carriers. This is the first successful clinical application of a synthetic lethal approach to targeting cancer. Exploitation of defects in DNA repair pathways through targeted inhibition of salvage repair pathways is an exciting anticancer approach, with potentially broad clinical applicability. Several PARP inhibitors are now in clinical development. This review outlines the biological function and rationale of targeting PARP, details pre-clinical and clinical data and discusses the promises and challenges involved in developing these antitumour agents. [Copyright &y& Elsevier]

Abstract: Human tumors frequently have defects in the maintenance of genomic integrity, which involve a loss of the appropriate response to DNA damage. These pathways of genome integrity include key proteins involved in cell cycle checkpoints, histone modifications, and DNA repair. In this review, we discuss opportunities for therapeutic intervention by exploiting these defects, with an emphasis on those processes which are primarily associated with the repair of double-strand breaks. As these defects are specific to tumor cells, the development of new anti-cancer agents targeting these pathways may have an enhanced therapeutic window, with limited normal tissue toxicity. [Copyright &y& Elsevier]

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5′-phosphate and 3′-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1−/− mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β (pol β) is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1−/−, and to a lesser extent in pol β−/− cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol β−/− cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1−/− cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.Cell Research (2008) 18:48–63. doi: 10.1038/cr.2008.7; published online 1 January 2008 [ABSTRACT FROM AUTHOR]

At least four disorders, ataxia telangiectasia (AT), an ataxia-telangiectasia-like disorder, early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1), and ataxia with oculomotor apraxia type 2, are accompanied by ocular motor apraxia (OMA), which is an impairment of saccadic eye movement initiation. The characteristic pathological findings of EAOH/AOA1 and AT are a severe loss of Purkinje cells, severe myelin pallor of the posterior columns, and moderate neuronal loss in the dorsal root ganglia and anterior horn. Purkinje cells stimulate the fastigial nucleus and suppress omnipause neurons to initiate saccadic eye movement. The selective loss of Purkinje cells might cause OMA and disturb the cancellation of the vestibulo-ocular reflex. These disorders have the following common clinical features: ataxia, involuntary movements, and peripheral neuronopathy. In addition, the causative genes for these disorders are associated with the DNA/RNA quality control system. The impairment of DNA/RNA integrity results in selective neuronal loss in these recessive-inherited ataxias. [ABSTRACT FROM AUTHOR]

Xrcc1 knockout embryos show increased DNA breakage and apoptosis in tissues of the embryo proper prior to death at embryonic day E6.5. An additional deficiency in Trp53 allows Xrcc1−/− embryos to enlarge slightly and initiate gastrulation although ultimately death is delayed by less than 24 h. Death presumably results from DNA damage that reaches toxic levels in the post-implantation mouse embryo. To investigate the level of XRCC1 protein needed for successful mouse development, we derived Xrcc1 transgene-complemented Xrcc1−/− mice that express Xrcc1 within the normal range or at a greatly reduced level (<10% normal). The greatly reduced XRCC1 protein level destabilized the XRCC1 partner protein DNA ligase III (LIG3) but still allowed for successful mouse development and healthy, fertile adults. Fibroblasts from these animals exhibited almost normal alkylation sensitivity measured by differential cytotoxicity. Thus, a large reduction of both XRCC1 and DNA ligase III has no observable effect on mouse embryogenesis and post-natal development, and no significant effect on cellular sensitivity to DNA alkylation. The presence of XRCC1, even at reduced levels of expression, is therefore capable of supporting mouse development and DNA repair. [Copyright &y& Elsevier]

Cellular DNA is inherently unstable, subject to both spontaneous hydrolysis and attack by a range of exogenous and endogenous chemicals as well as physical agents such as ionizing and ultraviolet radiation. For parasitic protists, where an inoculum of infectious parasites is typically small and natural infections are often chronic with low parasitemia, they are also vulnerable to DNA damaging agents arising from innate immune defenses. The majority of DNA damage consists of relatively minor changes to the primary structure of the DNA, such as base deamination, oxidation, or alkylation and scission of the phosphodiester backbone. Yet these small changes can have serious consequences, often being mutagenic or cytotoxic. Cells have therefore evolved efficient mechanisms to repair such damage, with base excision and single strand break repair playing the primary role here. In this chapter we describe a method for analyzing the activity from cell extracts of various enzymes involved in the base excision and single strand break repair pathways of trypanosomatid parasites.