Single Molecule Observations of Rad51 Nucleoprotein Filament Formation and Removal via Yeast Srs2

Abstract

The Rad51 in Saccharomyces cerevisiae plays a vital role in promoting homologous recombination (HR) by self-assembling into elongated nucleoprotein filaments on DNA and repair double-stranded DNA breaks (DSBs). However, mutations can arise from undesirable HR that lead to debilitating diseases such as rapid aging or breast cancer. The Srs2 helicase (a member of the SF1 helicase family) is an ATP dependent antirecombinase that disrupts the Rad51 presynaptic filament through physical interaction with the Rad51 recombinase.We are investigating the dynamics of individual Rad51 nucleoprotein filament on DNA and their interactions with Srs2 using ensemble and single molecule fluorescence microscopy. Our ensemble ATPase measurement shows a substantial increase in ATP hydrolysis when Srs2 is introduced in the presence of preformed Rad51 filament while fluorescence studies indicate that Srs2 efficiently removed Rad51 filaments. Unexpectedly, the burst of ATPase activity was not dependent on Srs2 concentrations applied, revealing a substoichiometric relationship between Rad51 and Srs2. Using single-molecule FRET assay, we demonstrate real time dynamics of individual Rad51 nucleoprotein filament and its interaction with Srs2. Our preliminary data suggests a possible mechanism by which Srs2 removes Rad51 nucleoprotein filaments.