Coordination of genome replication and anaphase entry by rDNA copy number inS. cerevisiae

Abstract

Ribosomal DNA (rDNA) copy number varies widely among individuals in many species, but the phenotypic consequences of such copy number fluctuations remain largely unexplored. In the yeast Saccharomyces cerevisiae, each rDNA repeat contains an origin of replication. Previous studies have demonstrated that the yeast rDNA locus can be a significant competitor for replication resources, suggesting that rDNA copy number variation may affect timely completion of genome-wide replication. We hypothesized that reduction in rDNA copy number and thus rDNA replication origins would reduce competition from the rDNA locus and thereby improve non-rDNA genome replication. To test this hypothesis, we engineered yeast strains with short rDNA arrays of 35 copies, a minimal copy number that still maintains wild type level ribosome function. Contrary to our hypothesis, the minimal rDNA strain displayed classic replication defects: decreased plasmid maintenance, delayed completion of chromosomal replication, and increased sensitivity to replication stress agonists. Although a normal rDNA array replicates late in S phase, the minimal rDNA array initiated replication in early S phase, resulting in delayed replication across the non-rDNA portions of the genome. Moreover, we discovered that absence of the rDNA fork barrier protein Fob1p increased DNA damage sensitivity in strains with early replicating rDNA. We present evidence that this increased sensitivity may be due to compromised regulation of cyclin phosphatase Cdc14p and premature entry into anaphase. Our results indicate that precocious rDNA replication, rather than total number of rDNA origins, compromises replication of the genome. Taken together, we suggest that the rDNA's large, late-replicating state is evolutionarily conserved to promote genome stability through timely genome replication and coordination of S phase completion with anaphase entry.

rDNA array length is a major determinant of replicative lifespan in budding yeast

“…S9 ). There was no significant difference between the strains with variable rDNA CN, which is in agreement with previous results ( 33 ). Taken together, our results suggest that the predominant underlying effect of higher rDNA CN on lifespan is an increase in SIR2 expression and reduced accumulation of ERCs.…”

“…Since yeast cells are known to maintain rRNA expression constant despite differences in rDNA CN ( 33 ), we wanted to examine whether this was true in the strains used here. RNA-Seq on total RNA (without enriching for mRNA) was performed to measure the levels of rRNA relative to the rest of the transcriptome ( SI Appendix , Fig.…”