Genome-wide mapping of DNA re-replication in S. cerevisiae

To maintain genomic stability, re-initiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6 and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that re-initiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location fr om re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated. Keywords: comparative genomic hybridization (CGH), DNA replication, re-replication Genomic DNA was purified from non-replicating and (re-)replicating cells, differentially labeled with Cy3 and Cy5, and competitively hybridized to a spotted microarray containing ORF and intergenic PCR products. Cy5/Cy3 ratios are normalized so that the average ratio of all elements equals the DNA content of the cells (as determined by flow cytometry). For most experiments, two hybridizations were performed from each of two independent genomic DNA preparations. (In some cases, two hybridizations were performed from a single genomic DNA preparation.) Experiments differ in the cell cycle position, whether re-replication was induced and combination of mutations in ORC, MCM and CDC6. Series contains a total of 50 hybridizations.