UCSF

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. Our lab had previously shown 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. 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 timing of origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we re-examined the issue of mechanistic redundancy and showed that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated demonstrating that the mechanisms blocking re-replication are overlapping rather than redundant.

Deregulation of these mechanisms leads to incomplete re-replication of the genome and has been shown to cause DNA damage checkpoint activation and double strand breaks. To understand the link between re-replication and DNA damage I performed a genome-wide kinetic analysis of fork progression using microarray CGH. I determined that fork progression was severely impaired during re-replication. During re-replication forks progressed at least five-fold slower than S phase forks and failed to complete duplication of the chromosomes. In addition, analysis of the progression of existing forks suggested that the forks were stalled or collapsed. I next investigated the potential source of this impairment and determined that impaired fork progression was not due to insufficient nucleotides. I also provided evidence that fork progression was impaired even when fork head-to-tail collisions due to multiple rounds of reinitiation were prevented. Thus this impairment is likely occurring at individual forks and is consistent with a model in which replication fork failure during re-replication leads to DNA lesions.