The origin recognition complex (Orc1-6) plays a fundamental role in the initiation of DNA replication by binding replication origins throughout the budding yeast cell cycle. ORC acts as a scaffold for the assembly of the pre-replicative complex (pre-RC) factors Cdc6, Cdt1 and a replicative helicase, the minichromosome maintenance (MCM2-7) complex in G1 phase. Upon assembly, origins are then said to be “licensed” for DNA replication. Previous models of pre-RC assembly and function have predicted that once MCMs have been loaded onto chromatin, ORC and Cdc6 are no longer required for DNA replication. In contrast, data from our lab strongly suggest a role for Orc6 in the maintenance of MCMs after pre-RC formation. Orc6 was found to be required for the chromatin maintenance of Mcm2 and more specifically, chromatin immunoprecipitation (ChIP) analysis demonstrated that Orc6 was necessary for the continued origin association of MCM proteins in late G1 at the early-firing origin ARS1, and the late-firing origin ARS609. It was also determined that after destabilization in the absence of Orc6, the pre-RC could be reassembled and facilitate DNA replication in late G1 after Orc6 expression had been turned back on. Interestingly, the clamp loading protein Cdc6 was also discovered to be essential to the maintenance of MCM proteins on bulk chromatin and at ARS1.

The Dbf4 protein is involved in the initiation of DNA replication, in complex with Cdc7 kinase, and also plays a role in the intra-S-phase checkpoint response via an interaction with Rad53 in Saccharomyces cerevisiae. The Dbf4 protein has three highly conserved motifs, called the N, M and C motifs. In view of the fact that a comprehensive analysis of the roles of the three motifs in the initiation of DNA replication and checkpoint response was not previously available, this study was, therefore, conducted. The objectives of the study were: (1) to assess the function of the three conserved motifs, with respect to their essentiality for cell viability, (2) to determine their roles in mediating interactions with other proteins (i.e. Cdc7, Orc2, Mcm2) involved in the initiation of DNA replication and with Rad53 in the intra-S-phase checkpoint response, and (3) to obtain the three-dimensional structure of the Dbf4 N-motif by X-ray crystallography. The Dbf4 N-motif was found to be nonessential for cell viability, mediates the interaction between Dbf4 and Rad53, and as well as the interaction with Orc2. A mutant lacking the N-motif (dbf4N), was found to have a growth defect and was hypersensitive to the genotoxic agents: hydroxyurea (HU) and methyl methane sulfonate (MMS), suggesting that a disruption in the intra-S-phase checkpoint occurred because of an abrogated Dbf4-Rad53 interaction. Double point mutation of two threonine residues of the N-motif (threonines 171 and 175) to alanines also caused an abrogated Dbf4-Rad53 FHA1 domain interaction. The Dbf4 M-motif was found to be essential for cell viability and mediates the interaction between Dbf4 and Mcm2. A single proline to leucine point mutation at amino acid residue 277 conferred resistance to HU and MMS and caused disrupted Dbf4-Mcm2 and Dbf4-Orc2 interaction, while Dbf4-Rad53 interaction was maintained. Thus, the alteration of the M-motif may facilitate the role of Dbf4 as a checkpoint target. The Dbf4-C motif was also found to be essential for cell viability. Deletion and point mutations to the C-motif affected the interactions between Dbf4 and Rad53, Orc2, Mcm2 and also with Mcm4. Attempts were also made to obtain the three-dimensional structure of Dbf4, using X-ray crystallography methods. The work presented here represents a thorough functional analysis of the three conserved domains of Dbf4 in Saccharomyces cerevisiae. These results can be used as a baseline for further research involving higher eukaryotic organisims, including humans. This is particularly of relevance in light of recent evidence demonstrating an overexpression of the human Dbf4 orthologue overexpression as a cancer phenotype in human cancer cells.

The origin recognition complex (Orc1-6) plays a fundamental role in the initiation of DNA replication by binding replication origins throughout the budding yeast cell cycle. ORC acts as a scaffold for the assembly of the pre-replicative complex (pre-RC) factors Cdc6, Cdt1 and a replicative helicase, the minichromosome maintenance (MCM2-7) complex in G1 phase. Upon assembly, origins are then said to be “licensed” for DNA replication. Previous models of pre-RC assembly and function have predicted that once MCMs have been loaded onto chromatin, ORC and Cdc6 are no longer required for DNA replication. In contrast, data from our lab strongly suggest a role for Orc6 in the maintenance of MCMs after pre-RC formation. Orc6 was found to be required for the chromatin maintenance of Mcm2 and more specifically, chromatin immunoprecipitation (ChIP) analysis demonstrated that Orc6 was necessary for the continued origin association of MCM proteins in late G1 at the early-firing origin ARS1, and the late-firing origin ARS609. It was also determined that after destabilization in the absence of Orc6, the pre-RC could be reassembled and facilitate DNA replication in late G1 after Orc6 expression had been turned back on. Interestingly, the clamp loading protein Cdc6 was also discovered to be essential to the maintenance of MCM proteins on bulk chromatin and at ARS1.

Maintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, 2002. / Includes bibliographical references (p. 157-158). Also available on the Internet.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 118-137). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 86-101). Also available in print.

Not available / text

No description available.

Replication fork stalling is a source of potentially tumourigenic genomic instability. The RecQ family helicase, Rqh1, is critical for the prevention of replication fork collapse and the formation of potentially deleterious recombination intermediates following fork stalling. Previous work in our lab with Schizosaccharomyces pombe (fission yeast) has shown that rqh10/rqh10 diploids are inherently unstable and show rapid reversion to the haploid state. The current work exploits this characteristic of fission yeast rqh10 mutants in a screen for genes that normally promote replication associated genomic instability. The rqh10rad30 mutant strains employed in this work incorporate the checkpoint deficiency caused by a lack of Rad3, so as to exacerbate the genomically unstable nature of this model. The current work describes the lithium acetate transformation based random mutagenesis by non-homologous integration of the ura4+ selectable marker into the rqh10rad30 fission yeast strains. This random mutagenesis generated extensive (24,500 – 50,000) mutant libraries. The quality of the libraries was assessed by can1 mutant assay, confirming an adequately extensive mutagenesis for the proposed screen. The process to be employed in the screen would involve the crossing of the mutant libraries, with the hope of generating diploids that will have two mutant copies of the same gene. Some of these diploids would appear unusually stable, showing a normal sporulation phenotype. This would indicate the mutation of a gene that normally promotes genomic instability following replication fork stalling. The practicality of the proposed screen of a vast number of diploids was assessed and described in detail in the current work. A technique involving inverse PCR (IPCR) adopted from previous work to identify mutants of interest, was also investigated. The investigation of this technique, and the work of others, suggests that transformation using such selectable marker fragments results in most apparent transformants containing extrachromosomal ura4+ fragments. These fragments are thought to provide the predominant template for IPCR, rendering the process unsuccessful at identifying the mutation in the current screen. However, with the mutant libraries generated, and the screen procedure detailed, the stage is set to conduct the screen once a more appropriate mutation location technique is identified. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-05-31 22:25:14.009