Single molecule characterization of the helicase Srs2 antirecombinase function

DE TULLIO LUISINA; KANIECKI KYLE; GREENE ERIC

Greensboro, GA

Congreso; Pew Biomedical Programs 2016 Annual Meeting; 2016

The Pew Charitable Trusts

Homologous recombination (HR) is a mechanism for the repair of double strand breaks (DSBs), which can arise from exposure to DNA damaging agents, such as ionizing radiation. DSBs can also occur due to replication fork collapse or through programmed events, such the Spo11-induced DSBs that form as a pre-requisite to recombination during meiosis. In the eukaryotes, HR requires the protein Rad51, which is an ATP-dependent ssDNA binding protein that forms long filaments on the processed ends of a DSB. This Rad51-ssDNA filament is the key intermediate in recombination and is necessary to catalyze the exchange of strands between homologous DNA molecules. HR is highly regulated because inappropriate recombination can result in gross chromosomal rearrangements, genetic disease and cancer. Srs2 is a key negative regulator of HR, and belongs to the superfamily (SF) 1 helicases, which use ATP hydrolysis to unwind DNA and remodel protein-DNA complexes1,2. Using total internal reflection fluorescence microcopy (TIRFM) and single-strand DNA (ssDNA) curtains3,4, we have established an assay that allows us to directly visualize Srs2 molecules as they translocate along single molecules of ssDNA. Our assays reveal that Srs2 can translocate in the 3?→5? direction at a mean velocity of ~150 nucleotides per second and can travel over distances spanning ≥20,000 nucleotides before falling of the ssDNA. While translocating, Srs2 strips Rad51 from the ssDNA, allowing Rad51 to be replaced by replication protein A (RPA), which is a eukaryotic ssDNA-binding protein that participates in all aspects of nucleic acid metabolism involving an ssDNA intermediate. This assay reflects the first direct visualization of Srs2 as it displaces Rad51 from ssDNA, and provides an experimental basis for establishing a more detailed understanding of the physical basis by which DNA translocases, such as Srs2 helicases, can regulate homologous recombination and help prevent the formation of inappropriate recombination intermediates. 1.Veaute, X. et al. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423, 309?312 (2003).2.Krejci, L. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305?309 (2003).3.Gibb, B. et al. Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules. Nat. Struct. Mol. Biol. 21, 893?900 (2014).4.Qi, Z. et al. DNA Sequence Alignment by Microhomology Sampling during Homologous Recombination. Cell 160, 856?869 (2015).