Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

Abstract

Summary Srs2 is a Super-Family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (~170 nucleotides per second) in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA, and in doing so promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

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

“…We have previously used double-tethered ssDNA curtains and total internal reflection fluorescence microscopy (TIRFM) to visualize the behaviors of Srs2 on RPA-coated ssDNA in the presence and absence of Rad52 (De Tullio et al, 2017). Here, we use single- and double-tethered ssDNA curtains to visualize the movement of Srs2 on Rad51-coated ssDNA (Figure 1A,1B & S2A).…”

“…Our data suggests that the C-terminus of Srs2 encompassing the Rad51 interaction domain is important for the initial recruitment of Srs2 to these clusters of RPA. Importantly, we have previously shown that this domain is also essential for efficient recruitment of Srs2 to RPA-coated ssDNA even when Rad51 is absent from the reactions (De Tullio et al, 2017). Given these results, we propose that Srs2 is directly recruited to the RPA clusters embedded between Rad51 filaments, rather than Rad51 itself.…”

“…An important implication of this finding is that as Srs2 begins stripping Rad51 from ssDNA, the newly created RPA-ssDNA that forms behind the leading Srs2 complex becomes available for the recruitment of additional Srs2 molecules (Figure 7). We have previously shown that Srs2 can strip RPA from the ssDNA, and new Srs2 recruitment events take place even more readily on the newly cleared naked ssDNA (De Tullio et al, 2017). Thus, the recruitment mechanism may consist of interactions with RPA, ssDNA, or both.…”

“…We propose that Srs2-dependent removal of Rad51 from the ssDNA lead to new tracts of RPA-ssDNA or near naked ssDNA, which can then function as loading sites for additional Srs2 molecules. Separate measurements indicate that Srs2 translocates on RPA-ssDNA at a rate that is ~20% faster than that on Rad51-ssDNA and travels even faster on naked ssDNA (De Tullio et al, 2017). The ability of Srs2 to translocate more rapidly on RPA-ssDNA would allow any newly loaded Srs2 molecules to quickly catch up to and merge with the leading Srs2 ensemble already present at the receding 3′ edge of the Rad51 filament.…”

Spontaneous self-segregation of Rad51 and Dmc1 DNA recombinases within mixed recombinase filaments

“…For all two-color images, we use a custom-built shuttering system to avoid the bleed through from the green into the red channel during image acquisition. With this system, images from the green (GFP) and the red (mCherry) channels are recorded independently, these recordings are offset by 100 milliseconds such that when one camera records the red channel image, the green laser is shuttered off, and vice versa (30)(31)(32).…”