Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52–ssDNA complexes

Abstract

Rad52 promotes the annealing of complementary strands of DNA bound by replication protein A (RPA) during discrete repair pathways. Here, we used a fluorescence resonance energy transfer (FRET) between two fluorescent dyes incorporated into DNA substrates to probe the mechanism by which human Rad52 (hRad52) interacts with and mediates annealing of ssDNA–hRPA complexes. Human Rad52 bound ssDNA or ssDNA–hRPA complex in two, concentration-dependent modes. At low hRad52 concentrations, ssDNA was wrapped around the circumference of the protein ring, while at higher protein concentrations, ssDNA was stretched between multiple hRad52 rings. Annealing by hRad52 occurred most efficiently when each complementary DNA strand or each ssDNA–hRPA complex was bound by hRad52 in a wrapped configuration, suggesting homology search and annealing occur via two hRad52–ssDNA complexes. In contrast to the wild type protein, hRad52RQK/AAA and hRad521–212 mutants with impaired ability to bind hRPA protein competed with hRPA for binding to ssDNA and failed to counteract hRPA-mediated duplex destabilization highlighting the importance of hRad52-hRPA interactions in promoting efficient DNA annealing.

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

“…In humans, RPA displacement by RAD51 is promoted instead by BRCA2 (Jensen et al 2010;; thus, there has been a specialization of function in human RAD52 compared with yeast Rad52, with the former likely reserved for SSA and second-end capture events. Like yeast Rad52, human RAD52 anneals complementary ssDNA strands coated with RPA (Grimme et al 2010). The mechanism of annealing involves the simultaneous interaction of ring-shaped RAD52 oligomers with two strands of ssDNA or RPA-ssDNA.…”

“…The mechanism of annealing involves the simultaneous interaction of ring-shaped RAD52 oligomers with two strands of ssDNA or RPA-ssDNA. Within these complexes, wrapping of ssDNA around the RAD52 rings induces a conformation of ssDNA that is favorable for annealing (Grimme et al 2010). RAD52-RPA protein -protein interactions and posttranslational modifications may also play important roles in the annealing of complementary RPA-ssDNA complexes.…”

Computational analysis of DNA repair pathways in breast cancer

“…Wilcoxon rank sum statistic (59,62). An interesting point to note is that the choice of gene-level statistic seems to have negligible effect on identifying significant pathways (59).…”

“…This list, however, is often inadequate in providing functional insights into the underlying mechanisms that drive the phenotypic distinction. To overcome this limitation, new methods termed pathway analysis have been developed, which shift the analysis from gene level to pathway level (56,(59)(60)(61)(62)(63)(64)(65)(66).…”

“…These approaches are sometimes also called "knowledgebased pathway analysis" [for example in (61)] to emphasise that they make use of prior biological knowledge about the pathways being studied. In many other articles, these approaches are referred to as gene set enrichment analysis (GSEA) or gene set analysis (GSA), especially when the sets of genes being analysed do not correspond to biological pathways [e.g., gene sets defined by gene ontology (GO) terms] (59,62,67,68).…”