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Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress.

ABSTRACT: Protein-protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein-protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein-protein interactions. We show that proteasome-mediated degradation of Polβ is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein-protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polβ in the cytosol via interaction between Polβ and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polβ with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polβ to the nuclear compartment and regulates the stability of Polβ via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polβ/NQO1 complex, enhancing the interaction of Polβ with XRCC1. Our results reveal that somatic mutations such as T304I in Polβ impact critical protein-protein interactions, altering the stability and sub-cellular localization of Polβ and providing mechanistic insight into how key protein-protein interactions regulate cellular responses to stress.

SUBMITTER: Fang Q

PROVIDER: S-EPMC6614843 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress.

Fang Qingming Q Andrews Joel J Sharma Nidhi N Wilk Anna A Clark Jennifer J Slyskova Jana J Koczor Christopher A CA Lans Hannes H Prakash Aishwarya A Sobol Robert W RW

Nucleic acids research 20190701 12

Protein-protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein-protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein-protein interactions. We show that proteasome-mediated degradation of Polβ is reg ...[more]

PMID: 31287140

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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

Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress.

Publications

Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress.

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