Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Interchromosomal Crossover in Human Cells Is Associated with Long Gene Conversion Tracts

ABSTRACT Crossovers have rarely been observed in specific association with interchromosomal gene conversion in mammalian cells. In this investigation two isogenic human B-lymphoblastoid cell lines, TI-112 and TSCER2, were used to select for I-SceI-induced gene conversions that restored function at the selectable thymidine kinase locus. Additionally, a haplotype linkage analysis methodology enabled the rigorous detection of all crossover-associated convertants, whether or not they exhibited loss of heterozygosity. This methodology also permitted characterization of conversion tract length and structure. In TI-112, gene conversion tracts were required to be complex in tract structure and at least 7.0 kb in order to be selectable. The results demonstrated that 85% (39/46) of TI-112 convertants extended more than 11.2 kb and 48% also exhibited a crossover, suggesting a mechanistic link between long tracts and crossover. In contrast, continuous tracts as short as 98 bp are selectable in TSCER2, although selectable gene conversion tracts could include a wide range of lengths. Indeed, only 16% (14/95) of TSCER2 convertants were crossover associated, further suggesting a link between long tracts and crossover. Overall, these results demonstrate that gene conversion tracts can be long in human cells and that crossovers are observable when long tracts are recoverable.

Sgs1 Regulates Gene Conversion Tract Lengths and Crossovers Independently of Its Helicase Activity

ABSTRACT RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3α, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Δ. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a “random walk,” and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Δ. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.

Multiple Heterologies Increase Mitotic Double-Strand Break-Induced Allelic Gene Conversion Tract Lengths in Yeast

Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at ∼100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised ∼7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.