Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein.
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 IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha 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 IIIalpha 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 x DNA ligase IIIalpha 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.
Beernink
Peter T
PT
Biosciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94551, USA. pbeernink@chori.org
Hwang
Mona
M
Ramirez
Melissa
M
Murphy
Michael B
MB
Doyle
Sharon A
SA
Thelen
Michael P
MP
D000911:Antibodies, Monoclonal / Q000737:chemistry; D002453:Cell Cycle; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D008958:Models, Molecular; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D012685:Sepharose / Q000737:chemistry; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D000076105:X-ray Repair Cross Complementing Protein 1
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D004247:DNA; D012685:Sepharose; D011065:Poly(ADP-ribose) Polymerases; D005978:Glutathione