Analysis of the human replication protein A:Rad52 complex: Evidence for crosstalk between RPA32, RPA70, Rad52 and DNA

Doba Jackson, Kajari Dhar, James K. Wahl, Marc S. Wold, Gloria E.O. Borgstahl

Research output: Contribution to journalArticle

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Abstract

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.

Original languageEnglish (US)
Pages (from-to)133-148
Number of pages16
JournalJournal of Molecular Biology
Volume321
Issue number1
DOIs
Publication statusPublished - Jan 1 2002

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Keywords

  • Double-strand break repair
  • Human Rad52
  • Protein-protein interaction
  • Replication protein A
  • Single-stranded DNA binding

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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