Mismatch recognition and subsequent processing have distinct effects on mitotic recombination intermediates and outcomes in yeast

Abstract

The post-replicative mismatch repair (MMR) system has anti-recombination activity that limits interactions between diverged sequences by recognizing mismatches in strand-exchange intermediates. In contrast to their equivalent roles during replication-error repair, mismatch recognition is more important for anti-recombination than subsequent mismatch processing. To obtain insight into this difference, ectopic substrates with 2% sequence divergence were used to examine mitotic recombination outcome (crossover or noncrossover; CO and NCO, respectively) and to infer molecular intermediates formed during double-strand break repair in Saccharomyces cerevisiae . Experiments were performed in an MMR-proficient strain, a strain with compromised mismatch-recognition activity ( msh6 Δ) and a strain that retained mismatch-recognition activity but was unable to process mismatches ( mlh1 Δ). While the loss of either mismatch binding or processing elevated the NCO frequency to a similar extent, CO events increased only when mismatch binding was compromised. The molecular features of NCOs, however, were altered in fundamentally different ways depending on whether mismatch binding or processing was eliminated. These data suggest a model in which mismatch recognition reverses strand-exchange intermediates prior to the initiation of end extension, while subsequent mismatch processing that is linked to end extension specifically destroys NCO intermediates that contain conflicting strand-discrimination signals for mismatch removal.

Recombinational repair of nuclease-generated mitotic double-strand breaks with different end structures in yeast

“…Recombination studies to date have used either HO or I-SceI, which each makes DSBs with 4-nt 3¢ overhangs. We previously used I-SceI to initiate HR between ectopic, 4.2 kb substrates (Guo et al 2017;Hum and Jinks-Robertson 2019) and a modified version of this system was used in the current study (see below). To create DSBs with 4-nt 5′ overhangs on the ends, I-SceI and its cleavage site were replaced by a zinc finger nuclease (ZFN) and its cleavage site.…”

“…The third, bidirectional category of NCOs contained hetDNA on each side of the DSB and is consistent with either Holliday junction dissolution or a double SDSA event. With the I-SceISNP8 substrates, 18% (27/149) of hetDNA tracts were bidirectional and among the unidirectional tracts, 82% (100/122) were downstream of the initiating DSB (Hum and Jinks-Robertson 2019). Moving the first SNP to 22 nt from the 3¢ end did not alter these distributions;…”

“…The break-proximal SNPs in the original I-SceI system (SJR3848) were 8 nt from the enzyme-created 3′ ends (SNP8) and frequently removed by Pol δ proofreading activity (Guo et al 2017;Hum and Jinks-Robertson 2019). The delitto perfetto method (Storici and Resnick 2006) was used to move the break-proximal SNPs to 22 nt from the 3′ ends (SNP22).…”

“…SJR4727 was then crossed with SJR3848 to derive SJR4748, an mlh1Δ strain containing the lys2::I-SceI recipient allele, the donor lys2Δ3′::I-SceIncSNP22-URA3-hisG allele, and galactose-inducible I-SceI. Finally, SJR4748 was crossed with SJR4258 (Hum and Jinks-Robertson 2019) to introduce a recipient allele with an upstream hisG marker (SJR4815).…”

Chromatin modifiers alter recombination between divergent DNA sequences

“…Such regulation, which can prevent deleterious chromosomal rearrangements, is initiated through the recognition of mismatches in heteroduplex DNA that form during strand invasion steps involving single-stranded DNA from a broken chromosome and a divergent duplex donor. In the yeast Saccharomyces cerevisiae, the Msh2-Msh6 and Msh2-Msh3 mismatch repair (MMR) complexes recognize mismatches in heteroduplex DNA and recruit the RecQ family helicasetopoisomerase complex Sgs1-Top3-Rmi1 to unwind the recombination intermediate in a process known as heteroduplex rejection (Datta et al 1996;Chen and Jinks-Robertson 1999;Nicholson et al 2000;Myung et al 2001;Spell and Jinks-Robertson 2004;Sugawara et al 2004;Goldfarb and Alani 2005;Hum and Jinks-Robertson 2019). If rejection does not occur, MSH complexes initiate repair of the mismatches in heteroduplex DNA after/while the break is repaired.…”