Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast

Abstract

Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.Recombination between sequences at identical (allelic) positions on homologous chromosomes is important for mitotic DNA repair and for meiotic chromosome disjunction. In addition to the normal allelic interactions, eukaryotic genomes contain large numbers of repeated sequences that can potentially serve as substrates for nonallelic (ectopic) recombination events. Ectopic recombination can be either reciprocal or nonreciprocal in nature and can lead to alterations in genome structure. Nonreciprocal recombination events (gene conversions), which involve the unidirectional transfer of information from one repeat to another, result in either the homogenization of repeats or the generation of novel sequences. Reciprocal recombination events (crossovers) involve the physical exchange of information between repeats and give rise to various types of genome rearrangements (e.g., duplications, deletions, inversions, and translocations). Given the genomedestabilizing effects of ectopic recombination, it is important that such events occur at low rates relative to allelic recombination.Two physical features of repeated sequences may be important for limiting potentially deleterious interactions (1): the total length of the repeats (substrate size) and the degree of sequence identity between the repeats (substrate homology). Studies in both prokaryotic systems (2, 3) and eukaryotic systems (4-8) have shown that substrate size does influence the efficiency of recombination. Similarly, studies done in a variety of organisms have shown that sequence divergence can be a potent barrier to homologous recombination (9-23). The molecular basis of this barrier is not entirely understood, but it is presumably related to the formation and͞or removal...

Heteroduplex rejection during single-strand annealing requires Sgs1 helicase and mismatch repair proteins Msh2 and Msh6 but not Pms1

“…The clear distinction between msh6⌬ and pms1⌬ in our study suggests that there could be additional steps in the assessment of heterology in the more complicated case where a single strand invades duplex DNA and where Pms1p clearly plays a role (12,30,31). One difference between our assays and those used in previous studies is that SSA occurs without the assistance of the strand exchange protein Rad51 (50,51), whereas HO-induced gene conversions primarily proceed by strand exchanges mediated by Rad51p (52-54).…”

“…In both vegetative and meiotic yeast cells, the presence of even a few mismatches can markedly reduce recombination (12,(25)(26)(27)(28)(29)(30)(31). These studies have also shown that mismatch repair mutations including msh2, msh6, pms1, and mlh1 can elevate the level of recombination events involving homeologous sequences (32,33).…”

“…msh2⌬, pms1⌬, and mlh1⌬ mutants are equally defective in mismatch correction during DNA replication and in repairing heteroduplex DNA in meiosis (22,23). Double mutants, such as pms1⌬ mlh1⌬ or mlh1⌬ msh2⌬, exhibit epistasis, implying that these genes work in the same repair pathway (24).In both vegetative and meiotic yeast cells, the presence of even a few mismatches can markedly reduce recombination (12,(25)(26)(27)(28)(29)(30)(31). These studies have also shown that mismatch repair mutations including msh2, msh6, pms1, and mlh1 can elevate the level of recombination events involving homeologous sequences (32,33).…”

Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity

“…This aspect of recombination surveillance is thought to be carried out, in part, by proteins involved in the mismatch repair system [47]. In yeast, a rapid drop-off in recombination frequency vs. percent divergence is observed for repetitive elements, wherein the first few mismatches have a more suppressive effect on recombination than the addition of further mismatches [48]. As a consequence of the relationship between recombination and divergence, the particular TE ecology of a host genome, including the number and age of copy numbers, will have a significant impact on the potential for ectopic instability.…”

Regulation of Recombination and Genomic Maintenance

“…Spies and Fishel (2015) provide a comprehensive discussion of heteroduplex rejection, the process by which mismatch repair enforces interaction between HR partners of sufficient homology. Already a single mismatch was found to reduce HR in a mismatch-repair-dependent fashion (Datta et al 1997). Use of the identical sister chromatid template in somatic cells allows setting a low threshold for heteroduplex rejection in mitotically growing cells.…”