title: Domain specific interaction in the XRCC1-DNA polymerase beta complex.
abstract: XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.
authors.affiliation: Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA.
authors.forename: A
authors.initials: A
authors.lastname: Marintchev
authors.forename: A
authors.initials: A
authors.lastname: Robertson
authors.forename: E K
authors.initials: EK
authors.lastname: Dimitriadis
authors.forename: R
authors.initials: R
authors.lastname: Prasad
authors.forename: S H
authors.initials: SH
authors.lastname: Wilson
authors.forename: G P
authors.initials: GP
authors.lastname: Mullen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004330:Drosophila; D004926:Escherichia coli; D005976:Glutaral; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C000619352:Xrcc1 protein, mouse; C000619372:Xrcc1 protein, rat; D019951:DNA Polymerase beta; D005976:Glutaral