The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair
2021; Elsevier BV; Volume: 297; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2021.101025
ISSN1083-351X
Autores Tópico(s)Cancer therapeutics and mechanisms
ResumoThe base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER. The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polβ/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polβ 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polβ/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein–protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polβ than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polβ/XRCC1 complex formation, polβ and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER. Base excision repair (BER) is a critical process for preventing the mutagenic and lethal consequences of DNA lesions such as apurinic/apyrimidinic (AP) sites and DNA base modifications arising from exposure to environmental hazards and various endogenous stressors (1Chatterjee N. Walker G.C. Mechanisms of DNA damage, repair and mutagenesis.Environ. Mol. Mutagen. 2017; 58: 235-263Crossref PubMed Scopus (448) Google Scholar, 2Krokan H.E. Nilsen H. Skorpen F. Otterlei M. Slupphaug G. Base excision repair of DNA in mammalian cells.FEBS Lett. 2000; 476: 73-77Crossref PubMed Scopus (301) Google Scholar, 3Lindahl T. Keynote: Past, present, and future aspects of base excision repair.Prog. Nucleic Acid Res. Mol. Biol. 2001; 68: xvii-xxxCrossref PubMed Google Scholar, 4Beard W.A. Horton J.K. Prasad R. Wilson S.H. Eukaryotic base excision repair: New approaches shine light on mechanism.Ann. Rev. Biochem. 2019; 88: 137-162Crossref PubMed Scopus (59) Google Scholar). 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For example, the studies have reported the disparate patterns for the localization of XRCC1 and its interacting partners to the sites of DNA damage and altered repair profiles of oxidative damage induced H2O2 in the XRCC1-deficient EM9 CHO cells expressing XRCC1 cancer-associated variants R194W, R280H, and R399Q (59Berquist B.R. Singh D.K. Fan J. Kim D. Gilenwater E. Kulkarni A. Bohr V.A. Ackerman E.J. Tomkinson A.E. Wilson D.M. Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population.Nucleic Acids Res. 2010; 38: 5023-5035Crossref PubMed Scopus (40) Google Scholar, 64Hanssen-Bauer A. Garten K. Gilijam K.M. Torseth K. Wilson D.M. Akbari M. Otterlei M. The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1.DNA Repair. 2012; 11: 357-366Crossref PubMed Scopus (26) Google Scholar). Although the significance of XRCC1 in the maintenance of genome integrity and cellular functionality has been well established, it remains less clearly defined at the biochemical level that how XRCC1 coordinates the BER steps through its scaffolding function during the substrate-product channeling process particularly at the downstream steps of BER pathway. Similarly, even though the significance of XRCC1 variants in the cellular functionality has been well defined, their biochemical characterization in the BER regulation through the coordinated interactions with the key repair enzymes polβ, ligase IIIα, APTX that play critical roles at the final steps of the coordinated repair is poorly understood. In the p
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