The Dbf4/Cdc7 kinase complex is required for the initiation of DNA replication and promotes this by acting upon members of the Mcm2-7 helicase. In addition to its role in replication, Dbf4/Cdc7 is a target of the S-phase checkpoint response through the Rad53 checkpoint kinase. In the budding yeast Saccharomyces cerevisiae, the regulatory subunit of this complex, Dbf4, is essential for kinase activity. Dbf4 is conserved throughout eukaryotes and contains three regions of discrete homology, termed the N, M, and C motifs, based on their location in the polypeptide chain. Motif C shows the highest conservation of all the motifs of Dbf4 and contains a CCHH type zinc finger. Mutation of the conserved cysteine and histidine residues of this zinc finger impair interactions with origin DNA and the Mcm2-7 helicase subunit Mcm2, but do not disrupt associations with Cdc7, Orc2, or Rad53. Cells where the endogenous Dbf4 CCHH zinc finger has been mutated exhibit slowed growth, and are delayed in their entry to, and progression through S-phase. These cells also display sensitivity upon long-term exposure to the ribonucleotide reductase inhibitor hydroxyurea (HU) and the DNA alkylating agent methyl methanesulfonate (MMS). The crystal structure of an amino-terminal region of Dbf4 containing motif N folds as a BRCA1-carboxy-terminal (BRCT) domain. This domain is required for the interaction with Rad53, but is not sufficient. A fragment of Dbf4 containing the BRCT domain and its fifteen preceding amino acids is sufficient to interact with Rad53 and folds as a modified BRCT domain containing an integral amino-terminal helical projection. Denoted the Helix-BRCT (HBRCT) domain, mutations that destabilize it abrogate the interaction with Rad53, and result in sensitivity to genotoxic agents. Dbf4 is recognized by the forkhead-associated FHA1 domain of Rad53, and the HBRCT domain of Dbf4 interacts directly with FHA1 in vitro. This interaction is phosphorylation independent and relies on a conserved lateral surface of FHA1, distinct from the phosphoepitope binding surface, which when mutated abrogates the interaction between Dbf4 and Rad53 and results in sensitivity to HU and MMS. The in vitro interaction between FHA1 and HBRCT does not require the ability of FHA1 to bind a phosphoepitope, while the in vivo interaction between full-length Rad53 and Dbf4 does. The FHA1 domain of Rad53 can simultaneously bind to a phosphopeptide and HBRCT, indicating that Rad53 recognition of Dbf4 may occur through a bipartite interaction using two surfaces of FHA1.

This thesis is a genetic study of the cytoplasmically- inherited determinant [psi] of Saccharomyces cerevisiae . [psi] is a potentiator of ochre suppression. The molecular basis of [psi] was investigated using mutagenesis as a probe. The psi⁺ phenotype (efficient suppression) can be mutated to psi^- phenotype (loss of suppression) by ultra-violet light (UV) and nitrosoguanidine (NTG) . The UV-induced mutation was a single-hit event and the pre-mutational lesion was partly photoreactivable . Repair or expression of UV-induced mutation to the [psi] determinant was under the same genetic control as for nuclear mutation. It was concluded that [psi] has a DNA genome. The 'extrachromosomal mutagens' thymidylate starvation, 5-fluorouracil, manganese chloride and cycloheximide failed to induce psi^- mutants whilst guanidine hydrochloride, dimethyl sulphoxide and potassium chloride were shown to induce this mutation at frequencies up to 100%. Several other physical and chemical agents caused a high frequency of loss of the psi⁺ phenotype. A new class of recessive nuclear mutation (pnm) was shown to cause a loss of the psi⁺ phenotype. A simple comple- mentation test was devised to distinguish them from cytoplasmic psi^- mutants. The dominant PNM^- mutation was shown not to cause a physical loss of the [psi] genome. Two mutants with a modified PNM^- phenotype were analysed. Attempts to demon- strate genetically the involvement of [psi] with the 80S ribosome were unsuccessful. The psi⁺ phenotype was conclusively demonstrated to be inherited independently' of the nucleus using a 'heterokaryon test'. Two models for the [psi] phenomena were proposed; one postulating the presence of a DMA 'plasmid' and one postulating the involvement of a stable, self-perpetuating metabolic state.

There are 30-35 copies of the retrotransposon Ty in the haploid genome of most laboratory strains of Saccharomyces cerevisiae. Ty elements are 5.9 kb long, have LTRs of 340 bp and produce a genomic RNA that is 5.7 kb in length, through which it transposes. They contain two ORFs; the TYA gene of the class I element Tyl-15 is 1,319 nucleotides long whilst its TYB gene, which possesses the consensus sequences of retroviral acid proteases, reverse transcriptases and integrases, is 3,984 nucleotides long. TYB overlaps TYA by 38 nucleotides at its 5' end and is expressed as a TYA;TYB fusion protein. In an attempt to asign some in vivo functions to the Ty encoded proteins, the whole transcriptional unit of Tyl-15 was overexpressed in yeast. Electron micrographs of the overexpressing cells revealed an abundance of cytoplasmic, ~60 nm virus-like particles (Ty-VLPs). These contain the 5.7 kb Ty RNA and have an associated reverse transcriptase activity. Their major protein is p2 (48 kD), which is a proteolytic derivative of the primary translation product of TYA, pi (50 kD), a protein that is also assembled into particles. A truncated pi, encoded by a TYA gene shortened by 60 codons at its 3' end, still aggregates into particles. Furthermore, the addition of α2-IFN (20 kD) onto its carboxy terminus, to produce a pl-IFN fusion protein of ~68 kD, does not interfere with its particle forming properties. The hybrid VLPs are easy to purify and elicit an antibody response in rabbits to the Ty and IFN epitopes. A series of fragments spanning Tyl-15 were inserted into the packaging analysis vector, pMA924. The association of the resulting PGK-Ty-IFN hybrid RNAs with Ty-VLPs, in the presence of abundant Ty encoded proteins, was analysed by Northern blotting and used to assess the packaging capabilities of Ty RNA. Unexpectedly, RNA sequences that, direct specific packaging are produced from regions throughout Tyl-15. The signals corresponding to TYA are probably located within two regions of the genomic RNA, 5' to nucleotide 236 and 3' to nucleotide 737; those corresponding to TYB are in at least two, currently undefined, areas. The TYB gene of the class II element Tyl-17 overlaps TYA by 44 nucleotides. The fusion of α2-IFN cDNA fragments into all three reading phases of TYB followed by a Western blot analysis demonstrated that, like the TYB gene of Tyl-15, it too is expressed as a TYA:TYB fusion protein.

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Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 101-120). Also available via World Wide Web.

Thesis (Ph.D.)-- University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999. / Errata slip inserted on back end-paper. Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999. Bibliography: leaves 106-125.

Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biologically Sciences. / Includes bibliographical references (leaves 100-105).

Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2005. / Dr Yury Chernoff, Committee Member ; Dr Jung Choi, Committee Member ; Dr Nick Hud, Committee Member ; Dr Roger Wartell, Committee Member ; Dr Harish Radhakrishna, Committee Member. Vita. Includes bibliographical references.