Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein

Abstract

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIα and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIα BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIα BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIα domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1·DNA ligase IIIα heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.