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No description available.

Cdt1 is a conserved and essential eukaryotic protein that is required for the licensing step of DNA replication. In order to control replication licensing and ensure a single round of DNA replication occurs per cell cycle, Cdt1 is subject to strict regulation. In Metazoa and S. pombe, Cdt1 is targeted for ubiquitylation and proteolysis in S phase and after DNA damage by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 is activated in Metazoa by an unusual mechanism that requires an interaction between the substrate and chromatin-loaded proliferating cell nuclear antigen (PCNA). This study addressed the involvement of PCNA in S. pombe Cdt1 proteolysis. A mutational analysis was undertaken to establish whether the Cdt1-PCNA interaction is conserved in S. pombe and the extent to which it regulates CRL4Cdt2-dependent turnover of the protein. S. pombe Cdt1 was shown to interact with PCNA in vivo and two variant PCNA-interacting peptide (PIP) motifs were identified in the protein. The two motifs function near-redundantly to promote both the Cdt1-PCNA interaction and the CRL4Cdt2-dependent proteolysis of Cdt1 in S phase and after DNA damage. The mutational analysis also resulted in the characterisation of two in-frame AUG codons in the cdt1+ reading frame. The second in-frame AUG codon was shown to be the principal initiator codon and was required to maintain wildtype Cdt1 protein levels and cell viability. CRL4Cdt2 is emerging as an important regulator of proteins that are involved in the control of cell cycle progression and the maintenance of genome stability. However, there are a number of outstanding questions regarding the mechanism and regulation of CRL4Cdt2. In order to address these questions, a genomics approach was taken to identify novel genes involved in Cdt1 regulation. A screen of non-essential S. pombe genes identified 17 candidate genes that, when inactivated, caused up-regulation of Cdt1. Unexpectedly, deletion of genes involved in homologous recombination resulted in a Rad3-dependent up-regulation of Cdt1. Further work is required to establish the biological significance of this finding.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2007. / Title from second page of PDF document. Includes bibliographical references (p. 91-102).

Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.