pSPObooster: A Plasmid System to Improve Sporulation Efficiency of Saccharomyces cerevisiae Lab Strains.

Common Saccharomyces cerevisiae lab yeast strains derived from S288C have meiotic defects and therefore are poor sporulators. Here, we developed a plasmid system containing corrected alleles of the MKT1 and RME1 genes to rescue the meiotic defects and show that standard BY4741 and BY4742 strains containing the plasmid display faster and more efficient sporulation. The plasmid, pSPObooster, can be maintained as an episome and easily cured or stably integrated into the genome at a single locus. We demonstrate the use of pSPObooster in low- and high-throughput yeast genetic manipulations and show that it can expedite both procedures without impacting strain behavior.

Two independent DNA repair pathways cause mutagenesis in template switching deficient Saccharomyces cerevisiae.

Upon DNA replication stress, cells utilize the postreplication repair pathway to repair single-stranded DNA and maintain genome integrity. Postreplication repair is divided into 2 branches: error-prone translesion synthesis, signaled by proliferating cell nuclear antigen (PCNA) monoubiquitination, and error-free template switching, signaled by PCNA polyubiquitination. In Saccharomyces cerevisiae, Rad5 is involved in both branches of repair during DNA replication stress. When the PCNA polyubiquitination function of Rad5 s disrupted, Rad5 recruits translesion synthesis polymerases to stalled replication forks, resulting in mutagenic repair. Details of how mutagenic repair is carried out, as well as the relationship between Rad5-mediated mutagenic repair and the canonical PCNA-mediated mutagenic repair, remain to be understood. We find that Rad5-mediated mutagenic repair requires the translesion synthesis polymerase ζ but does not require other yeast translesion polymerase activities. Furthermore, we show that Rad5-mediated mutagenic repair is independent of PCNA binding by Rev1 and so is separable from canonical mutagenic repair. In the absence of error-free template switching, both modes of mutagenic repair contribute additively to replication stress response in a replication timing-independent manner. Cellular contexts where error-free template switching is compromised are not simply laboratory phenomena, as we find that a natural variant in RAD5 is defective in PCNA polyubiquitination and therefore defective in error-free repair, resulting in Rad5- and PCNA-mediated mutagenic repair. Our results highlight the importance of Rad5 in regulating spontaneous mutagenesis and genetic diversity in S. cerevisiae through different modes of postreplication repair.