X-ray Repair Cross Complementing protein 1 (XRCC1) acts as a scaffolding protein in the converging base excision repair (BER) and single strand break repair (SSBR) pathways. XRCC1 also interacts with itself and rapidly accumulates at sites of DNA damage. XRCC1 can thus mediate the assembly of large multiprotein DNA repair complexes as well as facilitate the recruitment of DNA repair proteins to sites of DNA damage. Moreover, XRCC1 is present in constitutive DNA repair complexes, some of which associate with the replication machinery. Because of the critical role of XRCC1 in DNA repair, its common variants Arg194Trp, Arg280His and Arg399Gln have been extensively studied. However, the prevalence of these variants varies strongly in different populations, and their functional influence on DNA repair and disease remains elusive. Here we present the current knowledge about the role of XRCC1 and its variants in BER and human disease/cancer.

β-lapachone (β-lap), the NQO1 bioactivatable drug, is thought to be a promising anticancer agent. However, the toxic side effects of β-lap limit the drug use, highlighting the need for a thorough understanding of β-lap's mechanism of action. β-lap undergoes NQO1-dependent futile redox cycling, generating massive ROS and oxidative DNA lesions, leading to cell death. Thus, base excision repair (BER) pathway is an important resistance factor. XRCC1, a scaffolding component, plays a critical role in BER.

Previous studies investigating the association between X-ray repair cross-complementation group 1(XRCC1) polymorphisms and cervical cancer (CC) risk has provided inconsistent results. The aim of our study was to assess the association between the XRCC1 gene Arg399Gln, Arg194Trp, Arg280His polymorphisms and risk of CC.

X-ray repair cross complementing protein 1 (XRCC1) plays an important role in base excision DNA repair (BER) as a scaffolding protein for BER enzymes. BER is one of the basic DNA repair pathways repairing greater than 20,000 endogenous lesions per cell per day. Proper functioning of XRCC1, one of the most important players in BER, was suggested to be indispensable for effective DNA repair. Despite accumulating evidence of an important role that XRCC1 plays in maintaining genomic stability, the relationship between one of its most predominant variants, R280H (rs25489), and cancer prevalence remains ambiguous. In the current study we functionally characterized the effect of the R280H variant expression on immortal non-transformed mouse mammary epithelial C127 and human breast epithelial MCF10A cells. We found that expression of R280H results in increased focus formation in mouse C127 cells and induces cellular transformation in human MCF10A cells. Cells expressing R280H showed significantly increased levels of chromosomal aberrations and accumulate double strand breaks in the G1 cell cycle phase. Our results confirm a possible link between R280H and genomic instability and suggest that individuals carrying this mutation may be at increased risk of cancer development.

The X-ray repair cross-complementing gene (XRCC) family participates in DNA damage repair and its dysregulation is associated with the development and progression of a variety of cancers. However, XRCCs have not been systematically studied in non-small cell lung cancer (NSCLC). Using The Cancer Genome Atlas (TCGA) and Oncomine databases, we compared the expression levels of XRCCs between NSCLC and normal tissues and performed survival analysis using the data from TCGA. The correlations of XRCCs with the clinical parameters were then analyzed using UCSC Xena. Genetic alterations in XRCCs in NSCLC and their effects on the prognosis of patients were presented using cBioPortal. SurvivalMeth was used to explore the differentially methylated sites associated with NSCLC and their effect on prognosis. Next, the immunological correlations of XRCCs expression level were analyzed using TIMER 2.0. Finally, GeneMANIA was used to visualize and analyze the functionally relevant genes, while Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for functional and pathway enrichment analyses of prognostic genes. Our results revealed that XRCCs were overexpressed in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Univariate and multivariate Cox analyses showed that XRCC4/5/6 were independent risk factors for LUAD. Additionally, genetic alterations, methylation, and immune cell infiltration demonstrated an association between XRCC4/5/6 and poor prognosis in LUAD. Finally, the KEGG-enriched and non-homologous end-joining (NHEJ) pathways were shown to be associated with XRCC4/5/6. In conclusion, our study demonstrated that XRCC4/5/6 could be used as diagnostic and prognostic biomarkers for LUAD.

Recent studies have shown that tea polyphenols can cross the blood-brain barrier, inhibit apoptosis and play a neuroprotective role against cerebral ischemia. Furthermore, tea polyphenols can decrease DNA damage caused by free radicals. We hypothesized that tea polyphenols repair DNA damage and inhibit neuronal apoptosis during global cerebral ischemia/reperfusion. To test this hypothesis, we employed a rat model of global cerebral ischemia/reperfusion. We demonstrated that intraperitoneal injection of tea polyphenols immediately after reperfusion significantly reduced apoptosis in the hippocampal CA1 region; this effect started 6 hours following reperfusion. Immunohistochemical staining showed that tea polyphenols could reverse the ischemia/reperfusion-induced reduction in the expression of DNA repair proteins, X-ray repair cross-complementing protein 1 and apurinic/apyrimidinic endonuclease/redox factor-1 starting at 2 hours. Both effects lasted at least 72 hours. These experimental findings suggest that tea polyphenols promote DNA damage repair and protect against apoptosis in the brain.

Colorectal cancer (CRC) is one of the leading causes of cancer mortality and 5-Fluorouracil (5-FU) is the most common chemotherapy agent of CRC. A high level of X-ray repair cross complementing group 1 (XRCC1) in cancer cells has been associated with the drug resistance occurrence. Moreover, the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) has been indicated to regulate the cancer cell survival. Thus, this study was aimed to examine whether XRCC1 plays a role in the 5-FU/AMPK agonist (AICAR)-induced cytotoxic effect on CRC and the underlying mechanisms. Human HCT-116 colorectal cells were used in this study. It was shown that 5-FU increases the XRCC1 expression in HCT-116 cells and then affects the cell survival through CXCR4/Akt signaling. Moreover, 5-FU combined with AICAR further result in more survival inhibition in HCT-116 cells, accompanied with reduced CXCR4/Akt signaling activity and XRCC1 expression. These results elucidate the role and mechanism of XRCC1 in the drug resistance of HCT-116 cells to 5-FU. We also demonstrate the synergistic inhibitory effect of AMPK on 5-FU-inhibited HCT-116 cell survival under the 5-FU and AICAR co-treatment. Thus, our findings may provide a new notion for the future drug regimen incorporating 5-FU and AMPK agonists for the CRC treatment.

Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10-15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation. After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase beta, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM.

Epidemiologic studies have reported the association of X-ray repair cross-complementary group 1 (XRCC1) Arg399Gln polymorphisms with susceptibility to squamous cell carcinoma of the head and neck (HNSCC). However, the results were conflictive rather than conclusive. The purpose of this study was to clarify the association of XRCC1 Arg399Gln variants with HNSCC risk.

Oral cancer is the fourth most common type of cancer among males in Taiwan, and the prognosis for patients with advanced-stage oral squamous cell carcinoma (OSCC) remains poor. The present study investigated the prognostic value of three DNA repair genes, namely excision repair cross-complementing group 1 (ERCC1), ERCC2 and X-ray repair cross-complementing group 1 (XRCC1) in OSCC. The protein expression levels of XRCC1, ERCC1 and ERCC2 in oral cell lines were analyzed via western blotting and immunohistochemistry using samples from 98 patients with biopsy-proven OSCC, while the χ2 test was used to analyze the clinicopathological association. Kaplan-Meier estimates were used to determine the prognostic value of XRCC1, ERCC1 and ERCC2 for overall survival, and the log-rank test was used to evaluate the significance of differences. Multivariate analysis revealed a positive association between ERCC2 expression and OSCC recurrence (19.64-fold; 95% CI, 5.00-77.1; P<0.001). In addition, the high protein expression levels of XRCC1, ERCC1 and ERCC2 were associated with poor disease-free and overall survival rates. Therefore, the present study suggested that high ERCC2 expression may be a risk factor for OSCC recurrence.

The transcription factor Ets-1 (ETS proto-oncogene 1) shows low expression levels except in specific biological processes like haematopoiesis or angiogenesis. Elevated levels of expression are observed in tumor progression, resulting in Ets-1 being named an oncoprotein. It has recently been shown that Ets-1 interacts with two DNA repair enzymes, PARP-1 (poly(ADP-ribose) polymerase 1) and DNA-PK (DNA-dependent protein kinase), through two different domains and that these interactions play a role in cancer. Considering that Ets-1 can bind to distinctly different domains of two DNA repair enzymes, we hypothesized that the interaction can be transposed onto homologs of the respective domains. We have searched for sequence and structure homologs of the interacting ETS(Ets-1), BRCT(PARP-1) and SAP(DNA-PK) domains, and have identified several candidate binding pairs that are currently not annotated as such. Many of the Ets-1 partners are associated to DNA repair mechanisms. We have applied protein-protein docking to establish putative interaction poses and investigated these using centrality analyses at the protein residue level. Most of the identified poses are virtually similar to our recently established interaction model for Ets-1/PARP-1 and Ets-1/DNA-PK. Our work illustrates the potentially high number of interactors of Ets-1, in particular involved in DNA repair mechanisms, which shows the oncoprotein as a potential important regulator of the mechanism.

Transcription-coupled nucleotide excision repair factor Cockayne syndrome protein B (CSB) was suggested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated pathway to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fashion. Here we showed that recruitment of the preferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruitment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Remarkably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a specific transcription-dependent role for CSB in recruiting XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed genes, probably to allow XRCC1 recruitment to BER-intermediates masked by RNA polymerase II complexes stalled at these intermediates.

The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein-protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.

Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery.

The scaffold protein X-ray repair cross-complementing 1 (XRCC1) interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair (SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase (PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay (MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylation-dependent interaction site in XRCC1 and a forkhead-associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved Rev1-interacting region (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified high-affinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly.

Apurinic/apyrimidinic endonuclease-1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. Here, we show that APE1 is a target of the SIRTUIN1 (SIRT1) protein deacetylase. SIRT1 associates with APE1, and this association is increased with genotoxic stress. SIRT1 deacetylates APE1 in vitro and in vivo targeting lysines 6 and 7. Genotoxic insults stimulate lysine acetylation of APE1 which is antagonized by transcriptional upregulation of SIRT1. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress, and this vulnerability is rescued by overexpression of APE1. Activation of SIRT1 with resveratrol promotes binding of APE1 to the BER protein X-ray cross-complementing-1 (XRCC1), while inhibition of SIRT1 with nicotinamide (NAM) decreases this interaction. Genotoxic insult also increases binding of APE1 to XRCC1, and this increase is suppressed by NAM or knockdown of SIRT1. Finally, resveratrol increases APE activity in XRCC1-associated protein complexes, while NAM or knockdown of SIRT1 suppresses this DNA repair activity. These findings identify APE1 as a novel protein target of SIRT1, and suggest that SIRT1 plays a vital role in maintaining genomic integrity through regulation of the BER pathway.

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

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

The X-ray repair cross complementing 1 (XRCC1) protein is required for viability and efficient repair of DNA single-strand breaks (SSBs) in rodents. XRCC1-deficient mouse or hamster cells are hypersensitive to DNA damaging agents generating SSBs and display genetic instability after such DNA damage. The presence of certain polymorphisms in the human XRCC1 gene has been associated with altered cancer risk, but the role of XRCC1 in SSB repair (SSBR) in human cells is poorly defined. To elucidate this role, we used RNA interference to modulate XRCC1 protein levels in human cell lines. A reduction in XRCC1 protein levels resulted in decreased SSBR capacity as measured by the comet assay and intracellular NAD(P)H levels, hypersensitivity to the cell killing effects of the DNA damaging agents methyl methanesulfonate (MMS), hydrogen peroxide and ionizing radiation and enhanced formation of micronuclei following exposure to MMS. Lowered XRCC1 protein levels were also associated with a significant delay in S-phase progression after exposure to MMS. These data clearly demonstrate that XRCC1 is required for efficient SSBR and genomic stability in human cells.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), structural components [X-ray repair cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA-PKcs, DNA-PKcs/XRCC4, and DNA-PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB-inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA-PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA-PKcs. Moreover, ATM possesses overlapping functions with DNA-PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ-deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells.