Mechanism of single-stranded DNA annealing by RAD52-RPA complex.

RAD52 is important for the repair of DNA double-stranded breaks1,2, mitotic DNA synthesis3-5 and alternative telomere length maintenance6,7. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA)8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair10. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal11,12, and aberrant expression of RAD52 is associated with poor cancer prognosis13,14. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers15-17. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously18-20, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.

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.

Conformational adaptability of Redbeta during DNA annealing and implications for its structural relationship with Rad52.

Single-strand annealing proteins, such as Redbeta from lambda phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redbeta quaternary structure and Redbeta-DNA complexes. In the absence of DNA, Redbeta forms a shallow right-handed helix. The presence of single-stranded DNA (ssDNA) disrupts this structure. Upon addition of a second complementary ssDNA, annealing generates a left-handed helix that incorporates 14 Redbeta monomers per helical turn, with each Redbeta monomer annealing approximately 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redbeta monomers. Hence, we propose that Redbeta promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redbeta monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redbeta and the Rad52 families, which strengthens previous suggestions, based on similarities of their quaternary structures, that they share a common mode of action. Hence, our findings have implications for a common mechanism of DNA annealing mediated by single-strand annealing proteins including Rad52.