Two pathways drive meiotic chromosome axis assembly in<i>Saccharomyces cerevisiae</i>

In mitosis, sequences on sister chromatids are preferred as DNA repair templates, whereas in meiosis interhomolog-based repair is promoted. The switch of template preference during homologous recombinational (HR) repair of DNA breaks is a defining event in sexual reproduction. This preference is needed to establish linkages between homologous chromosomes that support meiotic chromosome segregation. In budding yeast, a central activity that enforces meiotic interhomolog bias is encoded in a meiosis-specific protein kinase complex, consisting of Red1, Hop1 and Mek1 (i.e., the RHM complex). Activation of Mek1 kinase in meiosis - dictated by complex formation and upstream DNA break-dependent signaling - leads to modification of HR factors and the establishment of interhomolog HR repair bias. How meiotic repair bias is established is a central question with implications for sexual reproduction, genetic diversity and genome stability. Studying the role of the RHM complex in DNA repair is complicated by the fact that Red1 and Hop1 are required for efficient meiotic DNA break formation. Here, we conditionally express RHM components in mitotically-dividing cells to show that these factors can autonomously establish the RHM complex outside of its physiological environment. In vivo analysis is complemented with in vitro biochemical reconstitution to analyze the composition of a Red1-Hop1 subcomplex. The RHM complex can be activated under DNA damaging conditions in mitotically-dividing cells, and activation depends on upstream Mec1 kinase function. We use this system to perform a structure-function analysis of RHM complex formation and Mek1 activation. Finally, we demonstrate that expressing active Mek1 in mitosis leads to rad51Δ-like DNA break sensitivity, suggesting that activation of the RHM complex is sufficient to reconstitute (parts of) its physiological function in mediating HR-based repair. This system should enable querying downstream effects of RHM complex action on DNA repair dynamics and template bias. Human homologs of Red1 and Hop1 are often aberrantly re-expressed in cancer cells. Our system has the potential to inform on (dys)functional effects of these genes on genome stability during human tumorigenesis.

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Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination inSaccharomyces cerevisiae

Shodhan

Xaver

Wheeler

et al. 2022

Preprint

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The DNA double strand breaks (DSBs) that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in budding yeast contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red are important for DSB formation; DSB levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with DSB levels. How axis protein levels influence DSB formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased DSBs and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in DSBs did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote DSB formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, only a small fraction of crossovers that formed at an insert locus required MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local DSB levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

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Two pathways drive meiotic chromosome axis assembly inSaccharomyces cerevisiae

Cited by 4 publications

References 69 publications

How do small chromosomes know they are small? Maximizing meiotic break formation on the shortest yeast chromosomes

How do small chromosomes know they are small? Maximizing meiotic break formation on the shortest yeast chromosomes

Studying meiosis in mitosis: Activating the meiosis-specific Red1-Hop1-Mek1 complex in mitotic budding yeast cells

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination inSaccharomyces cerevisiae

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