Human replication protein A-Rad52-single-stranded DNA complex: Stoichiometry and evidence for strand transfer regulation by phosphorylation

Xiaoyi Deng, Aishwarya Prakash, Kajari Dhar, Gilson S. Baia, Carol Kolar, Greg G. Oakley, Gloria E.O. Borgstahl

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)6633-6643
Number of pages11
JournalBiochemistry
Volume48
Issue number28
DOIs
StatePublished - Jul 21 2009

ASJC Scopus subject areas

  • Biochemistry

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    Deng, X., Prakash, A., Dhar, K., Baia, G. S., Kolar, C., Oakley, G. G., & Borgstahl, G. E. O. (2009). Human replication protein A-Rad52-single-stranded DNA complex: Stoichiometry and evidence for strand transfer regulation by phosphorylation. Biochemistry, 48(28), 6633-6643. https://doi.org/10.1021/bi900564k