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PMID: 10773072 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Domain specific interaction in the XRCC1-DNA polymerase beta complex.

Nucleic acids research ·Vol. 28 ·No. 10 ·2000-05-15 ·Pages 2049-59

Marintchev A, Robertson A, Dimitriadis EK, Prasad R, Wilson SH, Mullen GP

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.

MeSH Terms
Amino Acid Sequence Animals Cloning, Molecular Cricetinae Cross-Linking Reagents DNA Polymerase beta/chemistry,metabolism DNA Repair DNA-Binding Proteins/chemistry,metabolism Dimerization Drosophila Escherichia coli Glutaral Humans Mice Molecular Sequence Data Rats Recombinant Proteins/chemistry,metabolism Saccharomyces cerevisiae Sequence Alignment Sequence Homology, Amino Acid X-ray Repair Cross Complementing Protein 1
Chemicals
Cross-Linking Reagents DNA-Binding Proteins Recombinant Proteins X-ray Repair Cross Complementing Protein 1 XRCC1 protein, human Xrcc1 protein, mouse Xrcc1 protein, rat DNA Polymerase beta Glutaral
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Marintchev A
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA.
Robertson A
Dimitriadis E K
Prasad R
Wilson S H
Mullen G P
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/28.10.2049
Published
2000-05-15
Pages
2049-59
Language
English
Region
England
NLM ID
0411011
PMCID
PMC105377
Subset
IM
Grants
NIGMS NIH HHS · GM52738 · United States
External Links
PubMed source DOI: 10.1093/nar/28.10.2049 PMC Full Text View journal details
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