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Volume 12
Journal of Molecular and Genetic Medicine
ISSN: 1747-0862
May 21-23, 2018 Barcelona, Spain
&
Integrative Biology
6
th
International Conference on
Genomics and Molecular Biology
10
th
International Conference on
Genomics 2018 and Integrative Biology 2018
May 21-23, 2018
JOINT EVENT
Biophysical and biochemical insights into the mechanisms of action by Redβ during homologous
recombination
Sivaraman Subramaniam
Technische Universität Dresden, Germany
R
epair 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 λ phage Redβ. 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 Redβ monomers that weakly hold single DNA
strands together. Upon annealing, homodimerization of Redβ 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.
Redβ 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 β, 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 Redα-Redβ 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 Redβ and RAD52 SSAPs share ancestral and mechanistic roots.
sivaraman.subramaniam@tu-dresden.deJ Mol Genet Med 2018, Volume 12
DOI: 10.4172/1747-0862-C2-028