Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA.
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.
Jackson
Doba
D
Department of Chemistry, University of Toledo, 2801 West Bancroft Street, OH 43606-3390, USA.
Dhar
Kajari
K
Wahl
James K
JK
Wold
Marc S
MS
Borgstahl
Gloria E O
GE
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008027:Light; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D012542:Scattering, Radiation; D013539:Simian virus 40 / Q000235:genetics; D020349:Surface Plasmon Resonance
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497194:RAD52 protein, human; C497167:RPA1 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase