Identification of Mutations That Decrease the Stability of a Fragment of Saccharomyces cerevisiae Chromosome III Lacking Efficient Replicators

Abstract

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of $40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these ''originless'' chromosome fragments, we screened for mutants defective in the maintenance of an ''originless'' chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the ''originless'' fragment.

Rtt107 Is a Multi-functional Scaffold Supporting Replication Progression with Partner SUMO and Ubiquitin Ligases

“…3) lacking a portion of the right arm (referred to as Chr3ΔR, 170 kb) can be maintained in cells containing a normal Chr. 3 (Theis et al, 2007, 2010) (Figure 7A). One variant has 12 ARSs and the other has 7 ARSs, and are referred to as Chr3ΔR-12ARS and Chr3ΔR-7ARS, respectively.…”

“…One variant has 12 ARSs and the other has 7 ARSs, and are referred to as Chr3ΔR-12ARS and Chr3ΔR-7ARS, respectively. The extra five ARSs in the former are known to fire, and thus greatly reduce replicon sizes (Theis et al, 2007, 2010). Using PFGE coupled with Southern blot analysis, fully replicated Chr.…”

“…Chr3ΔR variants were introduced to cells using chromoduction as described in Theis et al, 2007, 2010. In brief, donor and recipient strains were grown to late log phase in SC-Leu-Trp media and YPD, respectively.…”

Reciprocal Chromosome Translocation Between the Left-End 220kb of Chromosome II and the Right-End 270kb of Chromosome X in Saccharomyces cerevisiae

“…The red/white colony color assay in the yeast S. cerevisiae has been reported as epistatic growth advantage of ade8-18, ade2 cells over red ade2 cells (Ugolini & Bruschi, 1996;Theis et al, 2007). That is, in the yeast S. cerevisiae, strain YNN290, the ade2-mutant, should be essentially shown as a red colony on YPD or SD-selected medium, but the red color of the colony should be reduced by the presence of the suppressor gene or the suppressive conditions against the ade2-mutant.…”

“…cerevisiae was a single eukaryotic cell, and each chromosome, non-coding regions and genes were small with few introns, and could be emerged of S. cerevisiae chromosomes. Therefore, the study using the system of S. cerevisiae might be straight-forward for the metabolic data and the heritable information to analyze to reflect the genome-level for the eukaryotic biological events (Schar, 2001;Kolodner et al, 2002;Theis et al, 2007). Moreover, the ade2-mutant in yeast S. cerevisiae also might be a heritable and the red/white colony color assay might an analytical tool for the distinction of the reciprocal chromosome translocation on genome-scale (Ugolini & Bruschi, 1996).…”