The scaffold protein XRCC1 stabilizes the formation of polβ/gap DNA and ligase IIIα/nick DNA complexes in base excision repair

Qun Tang,Melike Çağlayan

doi:10.1016/j.jbc.2021.101025

Abstract

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.

Highlights

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 [1,2,3,4]
The elution peak position of X-ray cross-complementing protein 1 (XRCC1) Nterminal DNA-binding (NTD) was at 17 ml, while the polβ/XRCC1 NTD protein complex was eluted at 14.4 ml
BER deficiency in mouse models and the defects in crucial repair proteins involving in short-patch BER have been associated with neurological disorders and cancer as shown in the functional studies with BER polymorphisms [68]

Summary

Introduction

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 [1,2,3,4]. We characterized XRCC1 with its BER protein partners (polβ, ligase IIIα, and APTX) to investigate XRCC1-mediated repair protein complex formation, protein–protein interactions, and DNA-binding affinity to the repair intermediates including gap and nick DNA with or without damaged ends.

Results

Conclusion