MutSγ-Induced DNA Conformational Changes Provide Insights into Its Role in Meiotic Recombination

Abstract

In many organisms, MutSg plays a role in meiotic recombination, facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans result in infertility and birth defects. To improve current understanding of MutSg function, this study investigates the binding affinities and structures of MutSg in complex with DNA substrates that model homologous recombination intermediates. For these studies, we overexpressed and isolated from Escherichia coli the yeast MutSg protein Saccharomyces cerevisiae (Sc) Msh4-Msh5. Sc Msh4-Msh5 binds Holliday junction (HJ)-like substrates, 3 0 overhangs, single-stranded (ss) forks, and the displacement loop with nanomolar affinity. The weakest binding affinities are detected for an intact duplex and open-junction construct. Similar to the human protein, Sc Msh4-Msh5 exhibits the highest affinity for the HJ with a K d < 0.4 nM in solution. Energy-transfer experiments further demonstrate that DNA structure is modulated by the binding interaction with the largest changes associated with substrates containing an ss end. Upon binding, Sc Msh4-Msh5 displaces the ss away from the duplex in most of the ss-containing intermediates, potentially enabling the binding of RPA and other proteins. In the case of the junction-like intermediates, Msh4-Msh5 binding either stabilizes the existing stacked structure or induces formation of the stacked X conformation. Significantly, we find that upon binding, Msh4-Msh5 stacks an open-junction construct to the same extent as the standard junction. Stabilization of the junction in the stacked conformation is generally refractory to branch migration, which is consistent with a potential role for MutSg to stabilize HJs and prevent branch migration until resolution by MutLg. The different binding modalities observed suggest that Msh4-Msh5 not only binds to and stabilizes stacked junctions but also participates in meiotic recombination before junction formation through the stabilization of single-end invasion intermediates.

A novel function for CDK2 activity at meiotic crossover sites

“…Following strand invasion, early recombination intermediates are thought to be stabilized by the MutSγ DNA clamp complex composed of MutS homolog proteins 4 and 5 (MSH4/MSH5) [ 46 , 47 , 64 ]. This stabilizing event is required both for the completion of homolog synapsis and also the downstream formation of meiotic COs. During this process, RAD51/DMC1 foci are typically replaced by MSH4 foci upon normal synapsis [ 7 , 46 , 65 ].…”

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization

“…A stable MutSg complex would be advantageous in a diploid background but may promote homeologous recombination in a neoallopolyploid. In S. cerevisiae, MutSg stabilizes single-end invasions as well as Holliday junction recombination intermediates (Lahiri et al, 2018). The ability of MutSg to stabilize early recombination intermediates comprising divergent heteroduplex sequences in wheat may have been negated by deletions in MSH5B and MSH4D.…”

Mutation of the ATPase Domain of MutS Homolog-5 (MSH5) Reveals a Requirement for a Functional MutSγ Complex for All Crossovers in Mammalian Meiosis

“…In vitro studies have demonstrated that the human and yeast MutSγ heterodimer can bind to D loops, HJs, single-stranded overhangs and other DNA substrates (Snowden et al 2004; Lahiri et al 2018). Binding to junctions enhances stability of these structures, while binding to single-stranded DNA promotes displacement of the overhang that could potentially allow for nucleoprotein filament formation involving, for example, RPA (Lahiri et al 2018).…”