An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase?
ABSTRACT 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.
- SourceAvailable from: Scott Maynard
[Show abstract] [Hide abstract]
- "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 . Briefly, frozen whole brain was thawed in 5 ml of ice-cold 1× MSH, pH 7.4. "
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 · 3.36 Impact Factor
[Show abstract] [Hide abstract]
- "LIG3 encodes ligase III, which is DNA-and ATPdependent , an essential component in the BER pathway in the repair of damages caused by free radicals  and, the levels of damage was found to be also directly associated with the expression levels of XRCC1  . "
ABSTRACT: Although tobacco and alcohol consumption are two common risk factors of head and neck cancer (HNC), other specific etiologic causes, such as viral infection and genetic susceptibility factors, remain to be understood. Human DNA is often damaged by numerous endogenous and exogenous mutagens or carcinogens, and genetic variants in interaction with environmental exposure to these agents may explain interindividual differences in HNC risk. Single nucleotide polymorphisms (SNPs) in genes involved in the DNA damage-repair response are reported to be risk factors for various cancer types, including HNC. Here, we reviewed epidemiological studies that have assessed the associations between HNC risk and SNPs in DNA repair genes involved in base-excision repair, nucleotide-excision repair, mismatch repair, double-strand break repair and direct reversion repair pathways. We found, however, that only a few SNPs in DNA repair genes were found to be associated with significantly increased or decreased risk of HNC, and, in most cases, the effects were moderate, depending upon locus-locus interactions among the risk SNPs in the pathways. We believe that, in the presence of exposure, additional pathway-based analyses of DNA repair genes derived from genome-wide association studies (GWASs) in HNC are needed.05/2013; 27(3):179-192. DOI:10.7555/JBR.27.20130034
[Show abstract] [Hide abstract]
- "The BER pathway is completed by a template-guided restoration of the damaged DNA strand by means of the sequential action of phosphodiesterases, DNA polymerases, and DNA ligases by either short-patch or FEN1- dependent long-patch repair (Frosina et al., 1996; Kim et al., 1998; Robertson et al., 2009). Additional accessory factors are important for coordination and regulation of the different steps of BER in vivo (Caldecott et al., 1994; Frosina et al., 1996; Kim et al., 1998; Kubota et al., 1996; Matsumoto et al., 1994). In addition to BER, epistasis analyses in Escherichia coli and yeast have revealed that nucleotide excision repair (NER) and recombination repair (RR) also contribute to cellular resistance to alkylating agents (Alseth et al., 2005; Memisoglu and Samson, 2000; Xiao and Chow, 1998). "
ABSTRACT: Modifications and loss of bases are frequent types of DNA lesions, often handled by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases, generating abasic (AP) sites that are subsequently cleaved by AP endonucleases, which further pass on nicked DNA to downstream DNA polymerases and ligases. The coordinated handover of cytotoxic intermediates between different BER enzymes is most likely facilitated by the DNA conformation. Here, we present the atomic structure of Schizosaccharomyces pombe Mag2 in complex with DNA to reveal an unexpected structural basis for nonenzymatic AP site recognition with an unflipped AP site. Two surface-exposed loops intercalate and widen the DNA minor groove to generate a DNA conformation previously only found in the mismatch repair MutS-DNA complex. Consequently, the molecular role of Mag2 appears to be AP site recognition and protection, while possibly facilitating damage signaling by structurally sculpting the DNA substrate.Structure 12/2012; 21(1). DOI:10.1016/j.str.2012.11.004 · 6.79 Impact Factor