Nishant, K. | bioRxiv Science

bioRxiv2018

The Neurospora crassa standard Oak Ridge background exhibits an atypically efficient meiotic silencing by unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) was discovered in crosses made in the standard Oak Ridge (OR) genetic background of Neurospora crassa. However, MSUD often was decidedly less efficient when the OR-derived MSUD tester strains were crossed with wild-isolated strains (W), which suggested either that sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was much less efficient in near-isogenic crosses made in a novel N. crassa B/S1 and the N. tetrasperma 85 genetic backgrounds. Possibly, regulatory cues that in other genetic backgrounds calibrate the MSUD response are missing from OR. The OR versus B/S1 difference appears to be determined by loci on chromosomes 1, 2, and 5. OR crosses heterozygous for a duplicated chromosome segment (Dp) have for long been known to exhibit an MSUD-dependent barren phenotype. However, inefficient MSUD in N. tetrasperma 85 made Dp-heterozygous crosses non-barren. This is germane to our earlier demonstration that Dps can act as dominant suppressors of repeat-induced point mutation (RIP). Occasionally, during ascospore partitioning rare asci contained >8 nuclei, and round ascospores dispersed less efficiently than spindle-shaped ones.\n\nGeneral abstractIn crosses made in the standard OR genetic background of Neurospora crassa, an RNAi-mediated process called MSUD efficiently silences any gene not properly paired with its homologue during meiosis. We found that MSUD was not as efficient in comparable crosses made in the N. crassa B/S1 and N. tetrasperma 85 backgrounds, suggesting that efficient MSUD is not necessarily the norm in Neurospora. Indeed, using OR strains for genetic studies probably fortuitously facilitated the discovery of MSUD and its suppressors. As few as three unlinked loci appear to underlie the OR versus B/S1 difference in MSUD.

genetics↗

bioRxiv2017

mlh3 separation of function and endonuclease defective mutants display an unexpected effect on meiotic recombination outcomes

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in bakers yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation of function class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for two mlh3 mutants with opposite separation of function phenotypes, and an endonuclease defective mutant. Unexpectedly, all three showed increases in the number of non-crossover events that were not observed in mlh3{Delta}. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.\n\nAuthor SummaryDuring meiosis, diploid germ cells that become eggs or sperm undergo a single round of DNA replication followed by two consecutive chromosomal divisions. The segregation of chromosomes at the first meiotic division is dependent in most organisms on at least one genetic exchange, or crossover event, between chromosome homologs. Homologs that do not receive a crossover frequently undergo non-disjunction at the first meiotic division, yielding gametes that lack chromosomes or contain additional copies. Such events have been linked to human disease and infertility. Recent studies suggest that the Mlh1-Mlh3 complex is an endonuclease that resolves recombination intermediates into crossovers. Interestingly, this complex also acts as a matchmaker in DNA mismatch repair (MMR) to remove DNA replication errors. How does one complex act in two different processes? We investigated this question by performing a mutational analysis of the bakers yeast Mlh3 protein. Five mutations were identified that disrupted MMR but not crossing over, and one mutation disrupted crossing over while maintaining MMR. Using a combination of biochemical and genetic analyses to further characterize these mutants we illustrate the importance of protein-protein interactions for Mlh1-Mlh3s activity. Importantly, we illustrate how defective meiotic components can alter the outcome of meiotic recombination events. They also provide new insights in our understanding of the basis of infertility syndromes.

genetics↗