Human replication protein A-Rad52-single-stranded DNA complex: stoichiometry and evidence for strand transfer regulation by phosphorylation.
The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential in DNA metabolism and is phosphorylated in response to DNA-damaging agents. Rad52 and RPA participate in the repair of double-stranded DNA breaks (DSBs). It is known that human RPA and Rad52 form a complex, but the molecular mass, stoichiometry, and exact role of this complex in DSB repair are unclear. In this study, absolute molecular masses of individual proteins and complexes were measured in solution using analytical size-exclusion chromatography coupled with multiangle light scattering, the protein species present in each purified fraction were verified via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western analyses, and the presence of biotinylated ssDNA in the complexes was verified by chemiluminescence detection. Then, employing UV cross-linking, the protein partner holding the ssDNA was identified. These data show that phosphorylated RPA promoted formation of a complex with monomeric Rad52 and caused the transfer of ssDNA from RPA to Rad52. This suggests that RPA phosphorylation may regulate the first steps of DSB repair and is necessary for the mediator function of Rad52.
Deng
Xiaoyi
X
The Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 987696 Nebraska Medical Center, Omaha, Nebraska 68198-7696, USA.
Prakash
Aishwarya
A
Dhar
Kajari
K
Baia
Gilson S
GS
Kolar
Carol
C
Oakley
Greg G
GG
Borgstahl
Gloria E O
GE
D002850:Chromatography, Gel; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008027:Light; D008954:Models, Biological; D010766:Phosphorylation; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism* / Q000648:ultrastructure; D051716:Replication Protein A / Q000378:metabolism* / Q000648:ultrastructure; D012542:Scattering, Radiation
D004277:DNA, Single-Stranded; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A