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.

The Rad52 protein has critical functions in distinct pathways of the homology-directed DNA repair, one of which is to promote the annealing of complementary strands of DNA. Both yeast and human Rad52 proteins organize into ring-shaped oligomers with the predominant form being a heptamer. Despite the wealth of information obtained in previous investigations, how Rad52 mediates homology search and annealing remains unclear. Here, we developed single-molecule fluorescence resonance energy transfer approaches to probe hRad52-mediated DNA annealing events in real time. We found that annealing proceeds in successive steps involving rearrangements of the ssDNA-hRad52 complex. Moreover, after initial pairing, further search for extended homology occurs without dissociation. This search process is driven by an interaction between 2 overlapping nucleoprotein complexes. In light of these observations we propose a model for hRad52-mediated DNA annealing where ssDNA release and dsDNA zippering are coordinated through successive rearrangement of overlapping nucleoprotein complexes.DNA recombination ͉ DNA repair ͉ fluorescence microscopy ͉ FRET ͉ single-molecule T he Rad52 protein belongs to a ubiquitous group of recombination mediator proteins whose function is essential for homologous recombination (HR), homology directed DNA repair, and rescue of collapsed replication forks, serving as a key player in the maintenance of genomic integrity (1-4). All characterized Rad52 proteins facilitate annealing of complementary DNA strands. Moreover, the annealing occurs even in the presence of single-strand binding protein RPA (1,(5)(6)(7)(8)(9). This function is critical during several important steps in the recombinational repair, including the second end capture, and the postinvasion ssDNA annealing within the D loop, and the synthesis-dependent strand annealing (7,10). Although the Rad52 mediator activity in mammalian cells overlaps with functions of other recombinational mediators (11-13), deciphering its molecular mechanism will provide insights into molecular basis of the cellular mechanisms responsible for accurate DNA repair.Cellular DNA transactions that require annealing of the 2 complementary DNA strands are conserved throughout biology. Consequently, all living organisms possess the ssDNA annealing proteins (14). Many of these proteins are of a different origin and are functional rather than structural homologues. Among them bacterial RecO (15) and invertebrate BRCA2 homologues [such as Ustilago maydis Brh2 (13)] were shown to facilitate annealing of ssDNA coated with their cognate ssDNA binding proteins. In contrast, Rad52 paralogue in budding yeast, Rad59 protein fails to facilitate annealing of ssDNA-RPA complexes, but can efficiently anneal naked DNA (16). Recent discovery that a bacteriophage Sak protein is both functional and structural homologue of eukaryotic Rad52 protein suggests the importance of not only annealing per se, but also of the mechanism by which the annealing proceeds (17).Loading of recombinase protei...

During homologous recombination and homology-directed repair of broken chromosomes, proteins that mediate and oppose recombination form dynamic complexes on damaged DNA. Quantitative analysis of these nucleoprotein assemblies requires a robust signal, which reports on the association of a recombination mediator with its substrate and on the state of substrate DNA within the complex. Eukaryotic Rad52 protein mediates recombination, repair, and restart of collapsed replication forks by facilitating replacement of ssDNA binding protein replication protein A (RPA) with Rad51 recombinase and by mediating annealing of two complementary DNA strands protected by RPA. The characteristic binding mode whereby ssDNA is wrapped around the Rad52 ring allowed us to develop robust and sensitive FRET-based assays for monitoring Rad52 interactions with protein-free DNA and ssDNA-RPA complexes. By reporting on the configuration of ssDNA dually labeled with Cy3 and Cy5 fluorescent dyes, solution-based FRET is used to analyze Rad52-RPA-DNA interactions under equilibrium binding conditions. Finally, FRET between Cy3 and Cy5 dyes incorporated into two homologous ssDNA molecules can be used to analyze interplay between Rad52-mediated DNA strand annealing and duplex DNA destabilization by RPA.

Reactive oxygen species (ROS) have been shown to play crucial roles in regulating various cellular functions, e.g. focal adhesion (FA) dynamics and cell migration upon growth factor stimulation. However, it is not clear how ROS are regulated at subcellular FA sites to impact cell migration. We have developed a biosensor capable of monitoring ROS production at FA sites in live cells with high sensitivity and specificity, utilizing fluorescence resonance energy transfer (FRET). The results revealed that platelet derived growth factor (PDGF) can induce ROS production at FA sites, which is mediated by Rac1 activation. In contrast, integrins, specifically integrin αvβ3, inhibits this local ROS production. The RhoA activity can mediate this inhibitory role of integrins in regulating ROS production. Therefore, PDGF and integrin αvβ3 coordinate to have an antagonistic effect in the ROS production at FA sites to regulate cell adhesion and migration.