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Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity.

ABSTRACT: Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase beta (Pol beta) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol beta have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol beta, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.

SUBMITTER: Cuneo MJ

PROVIDER: S-EPMC2872404 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity.

Cuneo Matthew J MJ London Robert E RE

Proceedings of the National Academy of Sciences of the United States of America 20100329 15

Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase beta (Pol beta) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol beta have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the int ...[more]

PMID: 20351257

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Project description: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.

Dataset Information

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity.

Publications

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase beta binding affinity.

Similar Datasets

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