An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase?

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808.
Molecular and Cellular Biology (Impact Factor: 4.78). 02/1994; 14(1):68-76. DOI: 10.1128/MCB.14.1.68
Source: PubMed


XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

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    • "Mitochondrial debris was removed by centrifugation at 16,200 × g for 10 min, and the supernatant was collected and stored at −80 • C. The protein concentration was measured using the Bradford method (BioRad). Mitochondria from mouse brain were isolated as described before [23]. Briefly, frozen whole brain was thawed in 5 ml of ice-cold 1× MSH, pH 7.4. "
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    ABSTRACT: Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.
    DNA repair 04/2014; 16(1):44–53. DOI:10.1016/j.dnarep.2014.01.015 · 3.11 Impact Factor
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    • "To understand how BRCT II and the phosphorylation function of XRCC1 affect the damage response of other proteins, we next examined how the repair proteins are recruited to SSBs. XRCC1 interacts with various proteins involved in SSBR: Polβ interacts with the N-terminus of XRCC1, and LigIIIα interacts with the BRCT II domain of XRCC1 (Caldecott et al., 1994; Marintchev et al., 2000). PNK and APTX interact with XRCC1 through the region between the BRCT I and BRCT II domains (Loizou et al., 2004; Luo et al., 2004). "
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    ABSTRACT: Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. The mechanisms explaining how XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 since 1) phosphorylation sites mutated in XRCC1 (X1-PM) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and 2) phosphorylation of XRCC1 is required for efficient polyubiquitination of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant up-regulation of ubiquitination, indicating that pARylation of XRCC1 prevents the poly-ubiquitination. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA, and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitination in live cells.
    Journal of Cell Science 07/2013; 126(19). DOI:10.1242/jcs.128272 · 5.43 Impact Factor
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    • "XRCC1 gene is an important component of the BER pathway encoding a scaffolding protein, which functions as a facilitator or coordinator in this pathway by directly interacting with poly (ADP-ribose) polymerase (PARP), DNA polymerase beta, and DNA ligase III [8], [9], [10]. Although numerous validated SNPs in XRCC1 gene have been identified in the dbSNP database (, "
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    ABSTRACT: Previous studies investigating the association between X-ray repair cross-complementing group 1 (XRCC1) polymorphisms and thyroid cancer risk have yielded inconsistent results. This meta-analysis was performed to derive a more precise estimation of the relationship between three XRCC1 polymorphisms and thyroid cancer risk. PubMed and EMBASE database were systematically searched to identify relevant studies. 10 publications were selected for this meta-analysis, including 11 studies for Arg399Gln polymorphism (1726 cases and 3774 controls), 7 studies for Arg194Trp polymorphism (1037 cases and 2487 controls) and 8 studies for Arg280His polymorphism (1432 cases and 3356 controls). The results in total population did not show any significant association between these three polymorphisms and the risk of DTC for all genetic models. However, when stratified by ethnicity, the results showed that Arg280His polymorphism was associated with an increased risk of DTC among Caucasians (Arg/His vs. Arg/Arg: OR = 1.45, 95% CI = 1.09-1.93; dominant model: OR = 1.43, 95% CI = 1.08-1.89; additive model: OR = 1.38, 95% CI = 1.05-1.80), whereas individuals carrying Arg/His genotype have a significantly reduced risk of DTC among Asians (Arg/His vs. Arg/Arg: OR = 0.71, 95% CI = 0.51-0.98). We also detected that 399Gln variant allele carriers might presented an overall decreased risk of DTC in mixed population. Furthermore, subgroup analyses by histological subtype revealed that Arg194Trp polymorphism was significantly associated with reduced risk for papillary thyroid carcinoma (PTC) (dominant model: OR = 0.71, 95% CI = 0.50-0.99). This meta-analysis suggests that Arg280His polymorphism might contribute to the susceptibility of DTC among Caucasians, whereas it might provide protective effects in Asians against the risk of DTC. Additionally, our results support the protective role of Arg194Trp polymorphism in developing PTC, and show evidence of an association between Arg399Gln polymorphism and decreased risk of DTC in mixed population.
    PLoS ONE 05/2013; 8(5):e64851. DOI:10.1371/journal.pone.0064851 · 3.23 Impact Factor
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