Abstract
Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2-Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2-Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51- and Dmc1-ssDNA in real time. We found that yeast Hop2-Mnd1 bound rapidly to Dmc1-ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2-Mnd1 with Rad51-ssDNA or RPA-ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2-Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR.
Highlights
Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material
Whereas during meiosis, programmed DSBs are initiated through the action of the universally conserved Spo11 enzyme [9, 10], HR proceeds through the resection of broken double-strand DNA, yielding long single-strand DNA overhangs that are paired with a homologous dsDNA elsewhere in the genome and used as a template for the repair of the damaged DNA [11,12,13]
Both findings are consistent with prior studies of Hed1 and Hop2–Mnd1 recombinase binding specificities [48, 49]. These findings suggest that during meiosis, when both Hed1 and Hop2–Mnd1 are present, each protein should associate with distinct subsections of the meiotic presynaptic complex: Hed1 should bind to the Rad51 subsections of the filaments, and Hop2–Mnd1 should bind to the Dmc1 subsections
Summary
Hop2–Mnd is essential during meiotic HR [59, 60, 62]. many of the basic biophysical properties of Hop2– Mnd binding to Dmc presynaptic filaments remain unknown. This observation suggests that the presence of Rad within these mixed recombinase filaments reduces the availability of potential binding sites for Hop2–Mnd1–mCherry Together, these results are most consistent with a model where Hop2–Mnd can bind to for molecules with spatially resolvable fluorescent foci, we measured the distance between adjacent Hop2– Mnd1–mCherry foci bound to presynaptic complexes reconstituted at 3:1 or 1:1 Rad51:Dmc ratios. These results are most consistent with a model where Hop2–Mnd can bind to for molecules with spatially resolvable fluorescent foci, we measured the distance between adjacent Hop2– Mnd1–mCherry foci bound to presynaptic complexes reconstituted at 3:1 or 1:1 Rad51:Dmc ratios This analysis revealed mean foci-to-foci distances of 1.2 Ϯ 0.54 and 1.5 Ϯ 0.49 m, respectively (Fig. 5C). These observations support the conclusions that Rad and Dmc form homotypic filaments and that Hop2–Mnd is recruited to Dmc, whereas Hed is recruited to Rad in the context of mixed meiotic filaments
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