Initiation of homologous recombination by Red beta and other single strand annealing proteins

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Single strand annealing proteins (SSAPs) promote homologous recombination and repair of double strand breaks. The best understood SSAP, RAD52, plays a central role in the maintenance of genome integrity in eukaryotes. A prominent prokaryotic SSAP, Red beta from lambda phage, is employed in the DNA engineering technology termed recombineering. Using bioinformatics tools and biochemical evaluations, in 2009 we published that these formerly disparate proteins were ancestrally related, thereby defining an SSAP superfamily and raising the prospect that SSAPs share a unifying mechanism. Despite the importance of RAD52, Red beta and other SSAPs, there is currently no proven mechanism for DNA annealing and the initiation of homologous recombination by any SSAP. Our recent publication on Red beta (Ander et al., 2015), which will be extended in the proposed research, includes a monomer-driven DNA clamping mechanism to initiate DNA annealing that stands in stark contrast to existing models based on initiation by the ~11mer protein rings formed by RAD52 and Red beta in vitro. In the proposed research, we aim to deepen the comparison between RAD52 and Red beta by (i) obtaining Red beta structures for comparison with the published RAD52 structure and (ii) pursue biochemical and single molecule comparisons of human RAD52, Lactococcal SakRad52 and Red beta. Additionally we aim to extend our understanding of SSAP-initiated homologous recombination by further defining the mechanism of Red recombination. Previously we showed that the extraordinary efficiency of Red recombination with double stranded DNA, which underlies its remarkable usefulness in recombineering, is due to single strand annealing onto the lagging strand template at the replication fork (Maresca et al., 2010). Red recombination with double stranded DNA also depends upon the protein-protein interaction between Red beta and its partner 5 to 3 exonuclease, Red alpha. We aim to (i) identify this interaction in detail and (ii) understand how the interaction contributes to recombination.