During DNA replication the DNA has to be unpacked, duplicated and repacked into chromatin, which is comprised of DNA and histone proteins (Nicholson and Muller, 2008b). The coordinated replication of DNA and histone protein synthesis is vital for the correct chromatin formation (Marzluff et al., 2008). The mechanism controlling the histone gene expression is only partially understood, especially the mechanism controlling any disturbances in the coordination of DNA replication and histone protein synthesis. Previous experiments suggested that the regulation mechanism of histone balance could involve regulation by a free histone protein pool (Takami and Nakayama, 1997b; Takami and Nakayama, 1997a; Dominski et al., 2005; Kroeger et al., 1995). A mathematical model was produced by Dr Hameister (Hameister, 2012) to describe the control of histone production during S phase. The parameters used were taken from literature. Modifications were made using experimentally measured data to replace literature values (Harris et al., 1991; Clark, 2006; Strachen and Read, 2003). The aim of the project was to both optimise the model by defining parameters to reflect what occurs naturally in the cell, as well as trying to validate the model. The DNA replication rate was measured by FACS analysis and was input into the model. Histone RNA levels during S phase were measured by Northern blots and histone RNA degradation rates were analysed. To confirm that the modified DNA replication rate was producing accurate simulations, the curve produced for the mRNA levels could be compared to the experimentally measured mRNA values (Figure 4.3.1). The over expression of an H2B gene was verified using Western and Northern analysis. The validation of the model that the histone gene balance was being regulated by a free histone pool was not absolutely confirmed. However, results seen in Figure 3.6.1, showed an increase in H2B RNA degradation in the sample with the additional gene due to the additional histone proteins. Further work is required for confirmation of the regulation mechanism.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xiii, 196 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical reference.

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Errors in DNA replication can cause chromosome instability and gross chromosomal rearrangements (GCRs). For my thesis work I investigate how chromosome instability can originate in the telomere. Here I report how defects in Cdc13, a telomere specific protein, lead to chromosome instability and GCRs in Saccharomyces cerevisiae. Using a temperature sensitive mutant of Cdc13, I find that cdc13-induced instability can be induced in a single cell cycle and synergizes with replication stress (dNTP depletion via hydroxyurea). Additionally, I find that Cdc13 has to be functional during the cell’s S phase to suppress chromosome instability. Further genetic analysis suggests that that cdc13-induced chromosome instability depends on the generation of single stranded (ss)DNA, but not on the activity of canonical double strand break (DSB) repair pathways such as homologous recombination or non-homologous end joining. Finally, I demonstrate that telomeric unstable chromosomes can later progress and trigger rearrangements at centromeric loci. This system, using the conditional nature of the cdc13 mutation, promises a more complex analysis of the ontogeny of chromosome instability: in this case from errors semi-conservative DNA replication through the telomere to the formation and resolution of unstable chromosomes.

The initiation of eukaryotic DNA replication occurs at replication origins, licensed by loaded double hexamers of Mcm2-7 proteins. The action of two kinases, DDK and S-CDK, triggers the loading onto Mcm2-7 of Cdc45 and the GINS complex to form the replicative CMG helicase. In yeast, the proteins Sld2 and Sld3 are CDK substrates that interact with Dpb11, and are required for the assembly of the CMG complex. Sld7 has been reported to associate with Sld3 throughout the cell cycle and regulate the function of Sld3 at replication initiation. Metazoan orthologues of these proteins have been identified: TopBP1, for Dpb11, is essential for replication;; Treslin, the Sld3 ortholog, is an essential S-CDK substrate that interacts with TopBP1 and is required for the CMG assembly;; in human cells MTBP has been found associated with Treslin, and is involved in the regulation of DNA replication, therefore MTBP as been proposed as a potential Sld7 ortholog. The aim of this project was to determine the role of MTBP in DNA replication using the Xenopus egg extract cell-free system. I found that MTBP and Treslin co-immunoprecipitate in metaphase and interphase extracts, suggesting that they form a complex throughout the cell cycle. MTBP, like Treslin, is recruited onto chromatin before the beginning of DNA replication and during S phase, independently of licensing and CDK/DDK activity. Immunodepletion of MTBP from extract leads to co- depletion of Treslin and causes a strong inhibition of DNA replication and impairment of CMG assembly. A partially purified fraction of extract, enriched for MTBP and Treslin, can rescue DNA replication in extracts depleted of MTBP. Analyses of the stoichiometry of the MTBP-Treslin complex has been carried out using gel filtration and sucrose gradient centrifugation; these identify a complex with an apparent molecular weight close to the one expected for a tetramer composed by two molecules of MTBP and two molecules of Treslin, resembling the model proposed for the interaction of the yeast proteins Sld3 and Sld7. These results, taken together, support the hypothesis that MTBP and Treslin form a multimeric complex that is required for the initiation of DNA replication and suggests that MTBP could be required to correctly position two Treslin molecules onto the MCM double hexamer to allow the bi-orientated firing of replication origins.

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