Human Replication Protein A−Rad52−Single-Stranded DNA Complex: Stoichiometry and Evidence for Strand Transfer Regulation by Phosphorylation

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. Double-strand breaks (DSBs) 1 in chromosomal DNA are efficiently repaired by homologous recombination (HR) (1). The RAD52 epistasis group of proteins, including Rad51, Rad52, and replication protein A (RPA), performs the repair of DSBs by HR (2, 3). Studies with Saccharomyces cerevisiae proteins (4-6) and with human proteins (7-10) demonstrated the importance of specific protein-protein contacts in controlling the functional interactions among Rad51, Rad52, and RPA. Rad52 acts as a mediator between RPA-coated ssDNA and Rad51 (11-13), and a similar function is also proposed for human BRCA2 protein (14). However, the molecular mechanisms for the delivery of Rad51 by mediators to RPA-coated ssDNA are not known in detail.RPA is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that is composed of three subunits that have been named for their molecular masses in kilodaltons as RPA70, RPA32, and RPA14 (15,16). The heterotrimer is the main biological form of RPA (17, 18) and is very stable when purified (19). RPA binds ssDNA more tightly than RNA or double-stranded DNA (dsDNA), prefers purines over pyrimidines (16), and has a 25-30-nucleotide footprint (20). RPA is composed of six oligonucleotide/oligosaccharide binding (OB) domains that bind ssDNA and/or participate in specific proteinprotein interactions (21). RPA is also known to undergo a significant conformational change upon binding DNA (22, 23) that appears to alter RPA's structure in a way that facilitates interactions with other proteins and ssDNA.The exact signal(s) for the presence of single-or doublestranded DNA breaks in a cell is still poorly understood, but the ssDNA-RPA complex appears to be an important component (24). Once DSBs are sensed, several downstream HR repair effector proteins are phosphorylated (25). RPA is phospho...

Posttranslational Modifications of Rad51 Protein and Its Direct Partners: Role and Effect on Homologous Recombination – Mediated DNA Repair

“…More recently, Shi and colleagues demonstrated by mutating the phosphorylation site of RPA that this posttranslational modification is required for Rad51 assembly (Shi et al, 2010). The importance of RPA phosphorylation during the presynaptic phase of HR was confirmed by Deng and colleagues who proposed a model in which RPA phosphorylation promotes Rad52 function and thus prepares DSB to be processed by Rad51 (Deng et al, 2009). Phosphorylation of the Rad52 mediator in a c-Abl dependant manner has also been described in response to ionizing treatment (Kitao and Yuan, 2002).…”

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…RAD52-RPA protein -protein interactions and posttranslational modifications may also play important roles in the annealing of complementary RPA-ssDNA complexes. For example, phosphorylated RPA, on interaction with RAD52, induces the handoff of ssDNA from RPA to RAD52 (Deng et al 2009). This suggests that there is a pathway for coupling the initiation of SSA or other annealing-dependent recombination processes to DNA damage signaling.…”

Human single-stranded DNA binding proteins: guardians of genome stability

“…If Rad52 is unable to release RPA from ssDNA and enable Rad51 exchange, the recombination would fail [74,75]. It has recently been reported that RPA phosphorylation can modulate HR repair [76][77][78], as the phosphorylation of RPA strengthens its interaction with Rad51 and Rad52 and is critical for Rad52-mediated Rad51 exchange and RPA dissociation from ssDNA [77]. This phenomenon is likely facilitated by the conformational change in phosphorylated RPA2.…”