Domain specific interaction in the XRCC1-DNA polymerase beta complex

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that form s complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-AD P-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain in teraction in the XRCC1-beta-Pol complex has provided information on the spe cificity and mechanism of binding, The domain structure of XRCC1, determine d using limited proteolysis, was found to include an N-terminal domain (NTD ), a central BIRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment an d the linker-BRCT-II C-terminal fragment were relatively stable to proteoly sis suggestive of a non-random conformation of the linker, A predicted inne r domain was found not to be stable to proteolysis, Using cross-linking exp eriments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa d omain, The XRCC1-NTD1-183 (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-NTD1-157 (residue s 1-157), The XRCC1-NTD1-183-beta-Pol 31 kDa domain complex was stable at h igh 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 b eta-Pol, the beta-Pol31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neit her the XRCC1-NTD1-159 nor the XRCC1(36-355) polypeptide was found to inter act with a beta-Pol thumbless polypeptide, A third XRCC1 polypeptide (resid ues 75-212) showed no Interaction with beta-Pol, In quantitative gel filtra tion and analytical ultracentrifugation experiments, the XRCC1-NTD1-183, wa s found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high a ffinity (K-d of 0.4-2.4 mu M). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD1-159 and beta-Pol that is o f an affinity comparable to other binding interactions involving beta-Pol.