Small but greedy: a minimal rDNA array outcompetes the genome for replication initiation

Abstract

Ribosomal DNA (rDNA) copy number varies widely among individuals in many species, but the phenotypic consequences of such copy number fluctuations remain relatively unexplored. Initiation of DNA replication has been detected within the rDNA, presumably required for maintenance of large rDNA arrays. Previous work in S. cerevisiae demonstrated that the rDNA locus—containing an origin of replication in each repeat—can be a significant competitor for replication resources, suggesting that rDNA copy number variation could affect timely completion of genome‐wide replication. To test the hypothesis that reduction in rDNA copy number (and thus rDNA replication origins) reduces competition from the rDNA locus and thereby improves non‐rDNA genome replication, we engineered an S. cerevisiae strain to have a short rDNA array of 35 copies, a minimal array that still maintains wild‐type level ribosome function. We compared this strain to a matched wild‐type strain with 180 rDNA copies. Contrary to our expectations, we found that the 35‐copy rDNA strain displayed classic replication defects: decreased plasmid maintenance, delayed completion of chromosomal replication, and increased sensitivity to replication stress agonists. The 35‐copy rDNA strain also exhibited delayed non‐rDNA origin firing and slower replication progression genome‐wide, reminiscent of mutants with hyperactive rDNA replication. We found that shrinking the rDNA locus dramatically alters rDNA replication time. In wild type cells, the majority of rDNA replicates in mid/late S‐phase of the cell cycle. When reduced to 35 copies, the rDNA locus becomes one of the earliest and most efficiently replicating regions of the genome, starting and completing replication 15 minutes ahead of the normally late‐replicating wild‐type rDNA locus. We therefore advocate that reduction of rDNA copy number alters genome replication through competition with an early‐replicating rDNA locus. Our results indicate that precocious rDNA replication, not total number of rDNA initiations, compromises replication of the genome and suggest that the conserved late‐replicating state of nucleoli may promote genome replication. Thus reductions in rDNA copy number may confer replication stress rather than replication relief, supporting an active role for rDNA copy number variation in determining stability of the genome.