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

Abstract

In mitosis, sister chromatids are preferred repair templates for homologous recombination, whereas in meiosis interhomolog-based repair is promoted. How this switch, which is a defining event in sexual reproduction, is accomplished remains poorly understood. In budding yeast, a meiosis-specific complex consisting of Red1, Hop1 and Mek1 (RHMc) enforces meiotic interhomolog bias, potentially through inhibition of intersister-based repair. The current data points to a linear assembly governing RHMc formation: the HORMA protein Hop1 associates with Red1 via a closure-motif-HORMA domain interaction, and Mek1 kinase is recruited through phospho-mediated interactions with Hop1. Here, via expression in mitotic cells we autonomously establish the RHM complex. In vivo analysis complemented with in vitro biochemical reconstitution shows that Mek1 associates with Red1, in a manner that might resemble binding of other kinases with scaffolding activators. The NH2-terminus of Red1 contributes to Hop1 binding, suggesting cooperative binding between Red1 and the HORMA domain of Hop1, beyond closure motif-based interactions. Meiotic activation of Mek1 kinase is dictated by complex formation and upstream DNA break-dependent signaling. We find Mek1 can be activated under DNA damaging conditions in mitotically dividing cells, where activation depends on upstream Mec1 kinase function and RHMc integrity. We perform a structure-function analysis of RHMc formation and Mek1 activation. Finally, we show that activation of Mek1 in mitosis leads to rad51{Delta}-like DNA break sensitivity, providing evidence for the model that RHMc instates meiotic interhomolog-based repair by inhibiting mitotic homologous recombination. Our analysis enables querying downstream effects of RHMc action on DNA repair. Because aberrant re-expression of homologs of Red1 and Hop1 leads to DNA repair defects in human cancer, our system can be used to study roles of these genes during tumorigenesis.