Genome-wide Analysis of Re-replication Reveals Inhibitory Controls that Target Multiple Stages of Replication Initiation

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-Replicative Complex (pre-RC) assembly outside of the G1 phase of the cell cycle. We have analyzed the effects of re-replication on the S. cerevisiae genome using a combination of Comparitive Genomic Hybridization (CGH) of re-replicating strains and Genome-Wide Location Analysis of pre-RC components. These data indicate which sites in the genome assemble pre-RCs under re-replication conditions, which sites undergo re-initiation and the extent of re-replication. Keywords: comparative genomic hybridization, ChIP-chip, re-replication, DNA replication, pre-RC We used strain SB1507 which contains a number of mutations that confer sensitivity to re-replication to monitor the effects of re-replication on the S. cerevisiae genome. Cells were induced to re-replicate with galactose and collected after either 45 minutes (for genome-wide location analysis) or three hours (for CGH). For comparision for pre-RC assembly, we also performed genome-wide location analysis for wild type cells during the G1 phase of the cell cycle. All of the above samples were hybridized to high-resolution, tiled DNA microarrays. We also showed that origin and origin-proximal DNA is sufficient to direct re-replication at an ectopic locus. For these experiments we used strains that had a re-replicating origin integrated at an endogenous locus. The strains were induced to re-replicate as described above for CGH. The resulting genomic DNAs were co-hybridized with G1-phase DNA to ORF DNA microarrays. Each experiment was performed with three experimental replicated per experiment.