TY - JOUR
T1 - Analysis of the human replication protein A:Rad52 complex
T2 - Evidence for crosstalk between RPA32, RPA70, Rad52 and DNA
AU - Jackson, Doba
AU - Dhar, Kajari
AU - Wahl, James K.
AU - Wold, Marc S.
AU - Borgstahl, Gloria E.O.
N1 - Funding Information:
We thank Ye Lao, Jeff Ohren, Wasantha Ranatunga and Andre Walther for providing purified proteins and technical assistance; D. Margaret Wheelock for antibody production; Dr Min Park for providing Rad52 expression vectors for wild-type Rad52, Rad52(1–192), Rad52(1–303) and Rad52(1–340); and Krishnamurthy Rajeswari for her initial work in developing the Rad52(218–418) construct. This work was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-98-1-8251 (to G.E.O.B.), DAMD17-00-1-0467 (to D.J.) and the National Institutes of Health RO1-GM44721 (to K.D. and M.W.).
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
KW - Double-strand break repair
KW - Human Rad52
KW - Protein-protein interaction
KW - Replication protein A
KW - Single-stranded DNA binding
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U2 - 10.1016/S0022-2836(02)00541-7
DO - 10.1016/S0022-2836(02)00541-7
M3 - Article
C2 - 12139939
AN - SCOPUS:0036349930
SN - 0022-2836
VL - 321
SP - 133
EP - 148
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -