Biophysical and biochemical insights into the mechanisms of action by Redβ during homologous recombination

Joint Event on 10^th International Conference on Genomics and Molecular Biology & 6^th International Conference on Integrative Biology

May 21-23, 2018 Barcelona, Spain

Sivaraman Subramaniam

Technische Universit�¤t Dresden, Germany

Posters & Accepted Abstracts: J Mol Genet Med

Abstract :

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]) such as eukaryotic RAD52 and �» phage Red�². These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. Using biophysical single molecule techniques, we have shown that homology is recognized by Red�² monomers that weakly hold single DNA strands together. Upon annealing, homodimerization of Red�² clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. Red�² clamp is characterized by a structural change and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, non-covalent, DNA-protein interaction. Using protein biochemistry and recombination assays, we have shown that C-terminally truncated Red �², whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Red�±-Red�² interaction, which requires not only contacts in the C-terminal domain but also at the N-terminus. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination, indicating two separable functions: one critical for dsDNA recombination and the other for recombination per se. These data further advance Red recombination model and show that Red�² and RAD52 SSAPs share ancestral and mechanistic roots. sivaraman.subramaniam@tu-dresden.de