Greenwood, Emma Jane; (2001) Functional analysis of the cell cycle control gene cdc18 in Schizosaccharomyces pombe. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Schizosaccharomyces pombe gene cdc18 has a role both in promoting DNA replication and initiating the checkpoint that acts to prevent mitosis until DNA replication has been successfully completed. It therefore plays an essential role in coordinating the cell cycle, thus maintaining genome integrity. I performed a functional analysis of cdc18 in order to understand its role throughout the cell cycle. I used different cdc18p constructs to discover which domains of the protein are required to carry out its functions. I examined this by assaying the ability of cells to re-replicate and block mitosis when the constructs were overexpressed. I discovered that the C-terminus of cdc18p is required for the re-replication phenotype and that this does not require a decrease in the activity of the mitotic kinase. The C-terminus is also required for the checkpoint function of cdc18p. The N-terminus of cdc18p can also block mitosis indirectly by binding to cdc2p. I then examined the Saccharomyces cerevisiae (ScCDC6), Xenopus laevis (XICDC6) and Homo sapiens (HsCDC6) cdcl8 homologues to determine their ability to complement the cdc18-K46 mutant. I also investigated whether they, like cdc18, are able to induce re-replication when overexpressed. None of the homologues was able to complement cdc18-K46, but overexpression of ScCDC6 was able to induce re-replication. Finally, I constructed a strain in which cdc18 was under the control of a repressible promoter. I made several site specific mutants of cdc18-the nucleotide triphosphate (NTP) binding and hydrolysis motif mutants, known as the Walker A (WA) and Walker B (WB) mutants, respectively, the putative nuclear localization signal mutants (NLS1, NLS2 and NLS1+NLS2) and the cdc2p phosphorylation site mutant (P1-6)-and introduced these at a different locus, where they were expressed under control of the cdc18 promoter. I investigated whether the mutants could proliferate in the absence of wildtype cdc18. Only the cdc18 WA and WB mutants failed to proliferate under these conditions. The WA mutant cannot initiate replication but is unable to inhibit mitosis, so undergoes an aberrant mitosis. The WB mutant is able to initiate, but not complete, replication and initiates a checkpoint signal. However, the checkpoint cannot be maintained and cells also enter an aberrant mitosis.

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