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DNA polymerase ? uses its lyase domain in a processive search for DNA damage.

ABSTRACT: DNA polymerase (Pol) ? maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol ? must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol ? are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol ? has evolved a mechanism for efficient DNA damage location. These assays revealed that Pol ? scans DNA using a processive hopping mechanism and has a mean search footprint of ?24 bp at predicted physiological ionic strength. Lysines within the lyase domain are required for processive searching, revealing a novel function for the lyase domain of Pol ?. Application of our processive search assay into nucleosome core particles revealed that Pol ? is not processive in the context of a nucleosome, and its single-turnover activity is reduced ?500-fold, as compared to free DNA. These data suggest that the repair footprint of Pol ? mainly resides within accessible regions of the genome and that these regions can be scanned for damage by Pol ?.

SUBMITTER: Howard MJ

PROVIDER: S-EPMC5397181 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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DNA polymerase β uses its lyase domain in a processive search for DNA damage.

Howard Michael J MJ Rodriguez Yesenia Y Wilson Samuel H SH

Nucleic acids research 20170401 7

DNA polymerase (Pol) β maintains genome fidelity by catalyzing DNA synthesis and removal of a reactive DNA repair intermediate during base excision repair (BER). Situated within the middle of the BER pathway, Pol β must efficiently locate its substrates before damage is exacerbated. The mechanisms of damage search and location by Pol β are largely unknown, but are critical for understanding the fundamental features of the BER pathway. We developed a processive search assay to determine if Pol β ...[more]

PMID: 28119421

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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

DNA polymerase ? uses its lyase domain in a processive search for DNA damage.

Publications

DNA polymerase β uses its lyase domain in a processive search for DNA damage.

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