Context-Dependent Remodeling of Rad51–DNA Complexes by Srs2 Is Mediated by a Specific Protein–Protein Interaction

Abstract

The yeast Srs2 helicase removes Rad51 nucleoprotein filaments from single-stranded DNA (ssDNA), preventing DNA strand invasion and exchange by homologous recombination. This activity requires a physical interaction between Srs2 and Rad51, which stimulates ATP turnover in the Rad51 nucleoprotein filament and causes dissociation of Rad51 from ssDNA. Srs2 also possesses a DNA unwinding activity and here we show that assembly of more than one Srs2 molecule on the 3' ssDNA overhang is required to initiate DNA unwinding. When Rad51 is bound on the double-stranded DNA, its interaction with Srs2 blocks the helicase (DNA unwinding) activity of Srs2. Thus, in different DNA contexts, the physical interaction of Rad51 with Srs2 can either stimulate or inhibit the remodeling functions of Srs2, providing a means for tailoring DNA strand exchange activities to enhance the fidelity of recombination.

Molecular Mechanism of Resolving Trinucleotide Repeat Hairpin by Helicases

“…Furthermore, the immobilized protein assay demonstrates that not Srs2, but a monomer of Sgs1 is sufficient for unwinding the DNA duplex. This is consistent with the previous finding that multimers of Srs2 are required for efficient unwinding (Qiu et al, 2013), with an unwinding concentration threshold (50nM), below which unwinding is limited (Lytle et al, 2014). …”

“…Here, we employed the full-length Srs2 protein instead of the C-terminal deletion mutant, Srs2 CΔ276 used in our previous study, although both have been shown to have similar helicase activities (Qiu et al, 2013). The concentration of Srs2 used here is comparable to previous studies (Anand et al, 2012; Bhattacharyya and Lahue, 2004) where we do not anticipate significant unwinding of the dsDNA (Lytle et al, 2014). When Srs2 (10nM) and ATP (1mM) was added to the pdT20 substrate, we observed a rapid FRET fluctuation between two FRET states (Figure 1B), consistent with the previously reported repetitive movement of Srs2 on single strand (ss) DNA, fueled by ATP hydrolysis (Qiu et al, 2013).…”

“…Sgs1 is capable of unwinding dsDNA immediately after the addition of a low concentration of protein (10nM) whereas Srs2, when applied at the same condition, exhibits repetitive movement on single strand DNA without unwinding the duplex DNA (Qiu et al, 2013). We note that Srs2, when applied at a much higher concentration (50-200nM), is capable of unwinding the same DNA efficiently in a tail length-dependent manner, albeit at a lower unwinding rate than Sgs1 (Lytle et al, 2014; Qiu et al, 2013). Furthermore, the immobilized protein assay demonstrates that not Srs2, but a monomer of Sgs1 is sufficient for unwinding the DNA duplex.…”

“…Taken together, we demonstrate that Sgs1, but not Srs2 can unwind duplex DNA as a monomer. This result combined with the requirement of 50-200nM of Srs2 for efficient dsDNA unwinding suggests that multimers of Srs2 is needed for DNA unwinding (Lytle et al, 2014; Qiu et al, 2013). …”

Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

“…In addition, recent studies have shown that E. coli UvrD translocates either as a monomer or as two tandem monomers, and that the tandem monomers are more processive than a single monomer (Comstock et al, 2015; Lee et al, 2013). Similarly, recent biochemical results have suggested that more than one Srs2 monomer is required for efficient DNA unwinding (Lytle et al, 2014). Our work now demonstrates that processive disruption of Rad51 filaments involves multiple Srs2 molecules acting upon the same filament end.…”