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ChIP-on-chip of Cdc45 in S. pombe

ABSTRACT: ChIP-on-chip of Cdc45 in synchronized cells operating with the analog-sensitive CDK fusion protein (Cdc13-Cdc2). Cells were released from a G2 block or from a G2 block followed by an extended G1 (G1+ 15 min extension). For the G2 block, samples were collected 30 minutes after release. For the G1+15 extension, samples were collected 3 minutes after release. Timepoint of collection was based on qPCR experiments showing Cdc45 binding at origins of replication.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE108453 | GEO | 2018/02/04

REPOSITORIES: GEO

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Project description:To achieve faithful replication of the genome once in each cell cycle, re-initiation of S-phase is prevented in G2 and origins are restricted from re-firing within S-phase. We have investigated the block to re-replication during G2 in fission yeast. The DNA synthesis that occurs when G2/M cyclin dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S-phase without mitosis but this has not been demonstrated to be the case. In a normal mitotic S-phase, the genome is uniformly replicated with no areas amplified relative to other regions, so there is an equal copy number of each portion of the genome. To determine whether equal rounds of replication or local amplification occurred in the cdc13 s/o strain we assayed genomic DNA from cells which had increased their DNA content to 16C-32C. Using microarrays, we measured the relative DNA content across the genome, using DNA from control G1-arrested cells as a reference. This experiment revealed that replication was essentially equal across the genome, with no region becoming significantly amplified to a higher copy number than any other. Since the genome was found to be essentially evenly replicated as would be expected if each round of replication corresponded to a normal S-phase, we investigated whether this was the result of a normal replication program at the level of origin firing. We asked whether S-phase origins are used to reduplicate the genome, and whether origins are used with the same efficiency as in wild type cells. We identified the origins utilized in the first endoreduplication cycle of cdc13 s/o using microarray analyses of cells treated with 11 mM HU. Samples were taken at 5 hours when most cells of the culture not treated with HU had undergone a doubling in DNA content. A total of 799 origins were identified, a number roughly similar to the 904 identified in a normal S-phase. We show here that on G2/M CDK depletion in G2, repeated S-phases are induced. Mostly normal mitotic S-phase origins were utilized although at different efficiencies, and replication was essentially equal across the genome. We conclude that CDK inhibits re-initiation of S-phase during G2, and if G2/M CDK is depleted replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.

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ChIP-on-chip of Cdc45 in S. pombe

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