Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

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.

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.

With the advances of technology and the popularization of mobile devices, the need of researching and discussing subjects related to mobile devices has raised. One of the subjects that needs to be further analyzed is data replication. This study investigates data replication on mobile devices focusing on power consumption. It presents four different scenarios that propose, describe, apply and evaluate data replication mechanisms, with the purpose of finding the best scenario that presents less energy consumption. In order to make the experiments, Sun SPOT was chosen as a mobile device. This device is fully programmed in a java environment. A different software was created in each scenario in order to verify the performance of the mobile devices regarding energy saving. The results found did not meet the expectations. While trying to find the best scenario a hardware limitation was found. Although software can be easily changed to fix errors, hardware cannot be changed as easily. The implications for the hardware limitation found in this study prevented the results to be optimal. The results found also imply that new hardware should be used in further experimentation. As this study proved to be limited, it suggests that additional studies should be carried out applying the new version of the hardware used in this study.

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 Tetrahymena thermophila rDNA minichromosome has been used as a model system for studying DNA replication. Previous studies have identified cis-acting replication determinants within the rDNA origin and promoter region including the type I element that is essential for replication initiation, fork progression and promoter activation. TIF1 is a non-ORC single strand-binding protein that binds the type I element in vivo. TIF1 binds opposing strands at the origin and promoter regions indicating that it may play a role in selectively marking these regions. In this dissertation, I use gene disruption to elucidate the role of TIF1 in replication. This work reveals that TIF1 represses rDNA origin firing, and is required for proper macronuclear S phase progression and division. Replication at the rDNA origin initiates precociously despite the observation that TIF1 mutants exhibit an elongated macronuclear S phase and a diminished rate of DNA replication. The amitotic macronucleus also displays delayed and abnormal division even though cells exit S phase with a wild-type macronuclear DNA content. Nuclear defects are also evident in the diploid micronucleus as TIF1 mutants contain fewer micronuclear chromosomes and are unable to pass genetic information to progeny. This defect is progressive as clonal mutant lines exhibit micronuclear instability during subsequent vegetative cell cycling. This work reveals that these macro- and micronuclear phenotypes may be the result of DNA damage as TIF1 mutants are hypersensitive to DNA damaging agents. This suggests that TIF1 mutants may have defects in the DNA damage response pathway. TIF1-deficient cells also incur DNA damage with no exogenous damaging agents. I propose that micro- and macronuclear defects witnessed in TIF1 mutant cells result from cells exiting S phase with compromised chromosomes due to the accumulation of DNA damage. Furthermore, TIF1 appears to play a role in the prevention, recognition or repair of DNA damage in addition to regulating rDNA replication and cell cycle progression and division. Additionally, TIF1 plays an essential role in the faithful propagation of both the macro- and micronuclear genomes.

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.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 103-117).