Pre-replicative complex (pre-RC) components the origin recognition complex (ORC), Cdc6, and Cdt1, play key roles in the recruitment, and loading of the replicative helicase, the minichromosome maintenance complex (Mcm2-7), onto DNA to license origins for replication. Until recently, the prevailing model for pre-RC assembly predicted that once MCMs are loaded at origins, ORC, Cdc6, and Cdt1 are dispensible for replication. Contrary to this model, previous work has shown that Orc6 is required following origin licensing, for the continued association of the MCM complex in late G1 phase. In this study, a similar role in pre-RC maintenance has been demonstrated for Cdc6, and Cdt1. Chromatin immunoprecipitation (ChIP) analysis has shown that late G1 phase depletion of either Cdc6, or Cdt1 leads to the destabilization of MCMs from origins, although this destabilization is more pronounced for Cdc6 depletion than for Cdt1. Furthermore, the resynthesis of Cdc6 following its depletion, allows for the reassembly of pre-RCs in late G1 phase, and restores competence for DNA replication. In this study, a potential role for Orc6 in mitosis/cytokinesis in budding yeast has also been characterized, as research with both Drosophila and human cell lines has pointed to a role for Orc6 in these processes. Deleting HOF1 and CYK3 (two proteins involved in cytokinesis in budding yeast) leads to a synthetic lethal phenotype, suggesting that the resulting gene products function in redundant cytokinetic pathways. Indeed, Hof1 has been shown to be primarily involved in actin ring contraction, while Cyk3 functions in septum formation, both pathways of which are important for budding yeast cytokinesis. Interestingly, previous work has identified an Orc6-Hof1 interaction in budding yeast. In this study, it has been demonstrated that following Orc6 depletion in a GAL1-ORC6/Δcyk3 strain, fluorescence activated cell sorting (FACS) analysis is consistent with a stronger cytokinetic defect phenotype than observed for Δcyk3 cells. Preliminary cell counts indicate that following Orc6 depletion, a higher percentage of GAL1-ORC6/Δcyk3 cells display misshapen mother bud necks than in an isogenic Δcyk3 strain. Cell synchronization experiments have demonstrated that Orc6 depletion during a G2/M phase arrest, leads to a block in cell cycle progression following release.

<p>RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.</p> / Dissertation

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Initiation of DNA replication. Department of Biochemistry and Molecular Genetics. Includes bibliographical references (142-171). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 139-153). Also available in electronic version. Access restricted to campus users.

DNA replication is initiated at replication origins, which are temporally regulated within the S phase of the cell cycle so that some origins initiate early, while others initiate late. S. cerevisiae telomeres of normal length replicate late during the S phase. However, shortened telomeres replicate early – coupling the regulation of length to the replication timing. The mechanism through which telomere length regulates the activation time of nearby replication origins is unknown. In this thesis, I find that Tel1, a homolog of human ATM kinase, is required for early replication of short telomeres. In the absence of Tel1, short telomeres of the yku70Δ mutant no longer replicate early. I tested the role of Histone H2A(X) phosphorylation at Serine-129, as the target of Tel1 kinase, in regulation of telomeric replication times. However, preventing this phosphorylation had only a minor effect, so H2A(X) is unlikely to be the sole Tel1 target in telomeric replication regulation. On the other hand, deletion of Rif1 – a telomeric length regulator – showed complete epistasis to tel1Δ in telomeric replication regulation, implying that Rif1 acts downstream of Tel1 in the regulation of telomeric replication. Proteomic analysis revealed Tel1-dependent phosphorylation of Rif1 upon telomere shortening, implicating Rif1 as a direct downstream target of Tel1. However, mutation of the Tel1-phosphorylation sites on Rif1 had only a small effect on telomere replication times, indicating that phosphorylation of Rif1 by Tel1 is not the sole mechanism governing replication timing of short telomeres.

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Thesis (Ph. D.)--University of Adelaide, Dept. Plant Science, 1997. / Errata pasted on front fly-leaf. Includes bibliographical references (leaves 139-173).

Thesis (Ph.D)--University of Wollongong, 2003. / Typescript. Includes bibliographical references: leaf 210-234.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 82-102). Also available in electronic version.