Base excision repair (BER) is the principal cellular defense against spontaneous hydrolytic, alkylation and oxidative damage to DNA, and corrects spontaneous and environmentally-induced mutagenic lesions involved in carcinogenesis. Thus, a molecular understanding of BER may lead to improvements in cancer prevention and cancer therapy, and have relevance to other diseases and aging. The objective of this proposal is to elucidate the precise biological function of XRCC1, one of at least five proteins required for proficient BER in human cells. The XRCC1 gene was cloned based on its ability to correct the repair defect of the hamster cell mutant EM9. XRCC1 protein was later shown to interact with several BER participants, namely DNA Ligase III, DNA Polymerase Beta, and Poly(ADP-Ribose) Polymerase. Complex formation with these proteins not only suggests a role for XRCC1 in mediating the final steps of BER, but suggests a function for XRCC1 in meiotic recombination. Still, the precise biochemical contribution of XRCC1 remains largely unknown. The researchers have developed methods to overexpress and purify recombinant XRCC1, and have found that XRCC1 possesses nuclease activity. These expression systems, as well as others to be generated, in combination with recently produced XRCC1-specific antibodies, will provide the means to characterize the exact role of XRCC1 in repair and recombination. Recombinant XRCC1 will be isolated from both bacteria and human cells, and the specificity of its nuclease activity determined on various damaged, nicked, and intact DNA substrates. To investigate a potential role in recombinational repair, the ability of XRCC1 to facilitate non-homologous DNA end joining will be tested. XRCC1-containing protein complexes isolated from mammalian cells will be examined for their functionality in repair assays and analyzed for the presence of the above XRCC1-interacting BER proteins, as well as novel complex partners. They will also look directly at possible interactions with other BER proteins (e.g. DNA glycosylases and the abasic endonuclease). Confocal immunofluorescence microscopy will be used to monitor in vivo localization patterns of several of the BER proteins following treatment with ionizing radiation and alkylating agents. Mutations in Xrcc-1 from mutant hamster cell lines will be sequenced, and these as well as other target mutations will be introduced into XRCC1 expression plasmids. The effects of these alterations on cellular repair capacity, in vitro biochemical activities and protein complex formation will be determined. Characterization of the participating proteins in BER, toward eventual reconstitution and molecular modeling of the process, will assist in efforts to understand those processes that maintain genomic integrity.
|Thornton, K H; Krishnan, V V; West, M G et al. (2001) Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha. Protein Expr Purif 21:401-11|
|Krishnan, V V; Thornton, K H; Thelen, M P et al. (2001) Solution structure and backbone dynamics of the human DNA ligase IIIalpha BRCT domain. Biochemistry 40:13158-66|
|Thornton, K; Forstner, M; Shen, M R et al. (1999) Purification, characterization, and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein. Protein Expr Purif 16:236-42|