The cell cycle is a highly orchestrated developmental process that eventually leads to the reproduction of a cell. In metazoans, it is driven by the successive activation of cyclin-dependent kinases (Cdk) and proper coordination of cell cycle transitions and processes ensure genomic stability. DNA replication takes place during S phase to faithfully duplicate a cell’s genetic material. In eukaryotes, S phase onset involves the initiation of numerous licensed replication origins across the genome and requires the activities of two protein kinases, S phase-Cdk and Cdc7. In this thesis, I present work relating to the role of the S phase-promoting kinases in DNA replication and S phase regulation. Using the cell-free system of Xenopus egg extracts, a small molecule inhibitor of Cdc7, PHA-767491, was characterised. PHA-767491 was then used to demonstrate that Cdc7 executes its activity early in S phase before the Cdk-dependent step. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike S-Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts, which rapidly reverses Cdc7-dependent phosphorylation of chromatin-bound Mcm4 and likely functionally lowers Cdc7 activity during an etoposide-induced checkpoint response. This provides a novel mechanism for regulating Cdc7 by counteracting its activity on essential replication substrates in the event of replicative stress. In the second part of the thesis, the design strategy for generating a Cdc7-conditional knockout mouse (cko) is outlined and results from the screen for a transgenic founder are presented. A Cdc7-cko mouse will be a valuable tool to further dissect Cdc7 function and regulation in mammalian cells. In the final section, S phase entry and progression in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2 was examined. Contrary to expectations, Cdk1/Cdk2 double knockout cells can enter S phase in the absence of detectable S phase-Cdk activity. S phase progression, however, was inefficient. Cdc6 and cyclin E1 proteins were found to accumulate in high levels in these cells. The exact function(s) and mechanism(s) for these observations remain to be discovered. With this work, I hope to provide additional insight into the roles and regulation of S phase kinases in eukaryotic DNA replication.

Intestinal homeostasis is governed by fate choices of stem cells residing in the intestinal crypt base. This must involve niche-specific co-ordination of cell division to guarantee that epithelial cells divide at the right time and place. These mechanisms operate to ensure precise control of the numbers of stem and differentiated cells. Little is known about how proliferative fate decisions are regulated in intestinal crypts. Both the placement of daughter cells within a particular niche, and their decision to enter and progress through the cell cycle, contribute. This thesis investigates the spatiotemporal control of cell division in intestinal crypts to understand the relationship between cell-cycle specific fate choices and intestinal homeostasis. Firstly, I describe a novel mode of asymmetric cell division within intestinal crypts. Using high resolution microscopy of intestinal organoids, I show that a subset of mitoses produce daughters that become displaced from one another after cytokinesis. This post-mitotic separation or the ‘positional asymmetry’ of daughter cells occurs in all cycling epithelial cells. These divisions may facilitate divergent fate of daughter cells and provides a general mechanism for stochastic niche exit. Post-mitotic separation is facilitated by interkinetic nuclear migration and selective tethering to the basement membrane during mitosis. Importantly, these mechanisms are altered in tissue carrying mutations in Adenomatous polyposis coli (Apc), highlighting its importance for normal tissue homeostasis. Secondly, I aimed to understand the dynamics of cell-cycle commitment in intestinal crypt compartments by investigating the DNA Replication Licensing System. The licensing system is a master regulator of proliferative fate in all cells in adult tissue. At its core is the regulated loading of the Mcm2-7 protein complex onto origins of replication exactly once per cell cycle. Engagement of the licensing system directly indicates commitment to proliferative cell fate. A technique to visualise licensing in intestinal crypts was developed. This revealed distinct proliferation zones in intestinal crypts. Mcm licensing was most prevalent in the lower transit-amplifying compartment, the zone enriched for early TA progenitors. Licensing is inhibited in terminally differentiated cells, and not detected in the transit-amplifying cells most proximal to the differentiated zone. Strikingly, the majority of ‘active’ intestinal stem cells were found in an unlicensed state. These data suggest that licensing decisions are delayed or inhibited until late G1 phase in intestinal stem cells and explains their longer cell-cycle. We postulate that this may provide a time window for niche cues to act, either stimulating cell-cycle entry or allowing retention in a ‘shallow’ G0 state. High resolution imaging of cell-cycle phases throughout the epithelium revealed remarkable cell-cycle co-ordination. This manifested in uninterrupted ‘ribbons’ of cells in similar cell-cycle states. This was due to lineage specific cell cycle co-ordination where adjacent daughter cells progress through the cell cycle at the same rate. These field effects are the result of co-ordinated cell-cycle progression between daughter cells. These observations were validated using living organoids expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI). These ribbons were occasionally interrupted by cells in other cell cycle phases suggesting the separation of sisters by daughters from another lineage. This suggests that cell-cycle coordination can facilitate post-mitotic separation, and influence stochastic niche exit.

One major pathway to overcome DNA damage induced replication blocks is translesion DNA synthesis, which is the replicative bypass of DNA damage by non-classical polymerases. For the cell to utilize translesion synthesis the non-classical DNA polymerase is recruited to sites of DNA damage, and a polymerase switch occurs between the stalled classical polymerase and the incoming non-classical polymerase. This process requires the replication accessory factor proliferating cell nuclear antigen (PCNA) and its monoubiquitination at Lys-164. To better understand the role of PCNA during translesion synthesis, I biochemically and structural characterized two PCNA mutant proteins, G178S and E113G PCNA, which are defective in translesion synthesis. The X-ray crystal structure of both mutant proteins showed a shift in an extended loop, called loop J, compared to the wild type PCNA structure. Steady state kinetic studies determined that in contrast to wild type PCNA which stimulates the non-classical polymerases, the two PCNA mutant proteins fail to stimulate the activity of the non-classical polymerase pol η. These results indicate that loop J in PCNA plays an essential role in facilitating translesion synthesis. During the structural studies of the E113G PCNA mutant protein I observed a unique PCNA structure that failed to form the characteristic PCNA ring shape structure, through traditional intersubunit interactions of domain A and domain B on neighboring subunits. Instead this non-trimeric PCNA structure formed A-A and B-B intersubunit interactions. The B-B interface is structurally similar to the A-B interface observed for the trimeric ring shaped form. In contrast the A-A interface is stabilized by hydrophobic interactions. The location of the E113G substitution is directly within this hydrophobic surface and would not be favorable in the wild type protein. This suggests that the side chain of Glu-113 promotes trimer formation by destabilizing these possible alternate subunit interactions. To biochemically and structurally characterize the impact of monoubiquitinating PCNA (Ub-PCNA), I developed an Ub-PCNA analog by splitting the protein into two self-assembling polypeptides. This analog supports cell growth and translesion synthesis in vivo, and steady state kinetic studies showed that the Ub-PCNA analog stimulates the catalytic activity of pol η in vitro. The X-ray crystal structure of Ub-PCNA showed that the ubiquitin moieties are located on the back face of PCNA. Surprisingly, the attachment of ubiquitin does not change PCNA's conformation. This implies that PCNA ubiquitination does not cause an allosteric change to PCNA, and instead facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for the non-classical polymerases.

All living organisms need to accurately replicate their genome to survive. Genomic replication occurs in three phases; initiation, elongation, and completion. While initiation and elongation have been extensively characterized, less is known about how replication completes. In Escherichia coli completion occurs at sites where two replication forks converge and is proposed to involve the transiently bypass of the forks, before the overlapping sequences are resected and joined. The reaction requires RecBCD, and involves several other gene products including RecG, ExoI, and SbcDC but can occur independent of recombination or RecA. While several proteins are known to be involved, how they promote this reaction and the intermediates that arise remain uncharacterized. In the first part of this work, I describe the construction of plasmid "mini-chromosomes" containing a bidirectional origin of replication that can be used to examine the intermediates and factors required for the completion reaction. I verify that these substrates can be used to study the completion reaction by demonstrating that these plasmids require completion enzymes to propagate in cells. The completion enzymes are required for plasmids containing two-replisomes, but not one replisome, indicating that the substrate these enzymes act upon in vivo is specifically created when two replication forks converge. Completion events in E. coli are localized to one of the six termination (ter) sequences within the 400-kb terminus region due to the autoregulated action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner. In the second part of this work, I examine how the presence of ter sequences affect completion on the 2-replisome plasmid. I show that addition of ter sequences modestly decreases the stability of the two-replisome plasmid and that this correlates with higher levels of abnormal, amplified molecules. The results support the idea that ter sites are not essential to completion of DNA replication; similar to what is seen on the chromosome. Rec-B-C-D forms a helicase-nuclease complex that, in addition to completion, is also required for double-strand break repair in E. coli. RecBCD activity is altered upon encountering specific DNA sequences, termed chi, in a manner that promotes crossovers during recombinational processes. In the third part of this work, I demonstrate that the presence of chi in a bidirectional plasmid model promotes the appearance of over-replicated linear molecules and that these products correlate with a reduced stability of the plasmid. The effect appears specific to plasmids containing two replisomes, as chi on the leading or lagging strand of plasmids containing one replisome had no-effect. The observation implies chi promotes a reaction that may encourage further synthesis during the completion reaction, and that at least on the mini-chromosomes substrates, this appears to be a destabilizing force.

As an Adenovirus infects a host cell a multitude of molecular interactions occur, some driven by the virus and some driven by the cell it is infecting. Many of these areas of Adenovirus biology have been intensely studied over the last half century, however, many questions remain unanswered. The aim of this study was to investigate, more closely, a long studied molecular interaction, namely the role of the tumour suppressor p53 in the Adenovirus life cycle, and also to investigate the related, but much less studied, interaction between Adenoviruses and the host cell DNA repair machinery. Controversy surrounds the role of p53 in the Adenovirus life cycle, with current dogma favouring the view that p53 is inactivated, as it presumably presents an obstacle to a productive infection. In Chapter 3, a standardised infection protocol was developed to examine this area of Adenovirus biology more closely. This was followed with an array of cell viability and western blotting analyses that not only showed p53 was not an antagonist of the Adenovirus life cycle, but in some cases p53 acted as a protagonist. Isogenic cell lines were used to reinforce this point. Following this, data were provided that virus DNA replication was linked to the ability of an Adenovirus to kill cells. Furthermore, p53 was shown by immunofluorescence to be present in infected cells at a time that corresponded with virus DNA replication, albeit at low levels. By adding p53 back into cells, it was shown that the number of Adenovirus progeny could be stimulated to levels produced in genetically wild type TP53 cells. A selection of promoter/reporter assays and infection/transfection assays then showed how p53 might be aiding the virus life cycle. These data showed that low levels of p53 cooperated with the Adenovirus transactivator, E1A, to promote late gene expression, and this translated into a modest increase in virus late antigens in infected cells. Taken together these data show that, contrary to current dogma, p53 generally aids an Adenovirus infection and it may do this through promoting virus late gene expression. Recent data have emerged suggesting Adenoviruses must disable the host DNA double-strand break machinery to achieve a productive infection. As this area of Adenovirus biology is in its infancy, and as p53 has recently been identified as an integral component of these DNA repair processes, the contributions of the host cell repair machinery to Adenovirus biology were examined in Chapters 4 and 5. In Chapter 4, western blotting showed that upon Adenovirus infection, a key component of the homologous recombination repair machinery, Rad51, was markedly up-regulated. This up-regulation occurred independently of other key repair proteins, and was found to be a generalised feature of an Adenovirus infection. Surprisingly, p53 did not appear to be involved in this up-regulation, and neither were several other nodal host regulatory proteins. The up-regulation was then linked to Adenovirus DNA replication using a temperature-sensitive mutant Adenovirus, ts125. In Chapter 5, functional analysis of this up-regulated protein showed that Rad51 colocalised with Adenovirus replication centres. This colocalisation coincided with a time when virus DNA replication was occurring. Furthermore, transient over-expression of Rad51 drastically increased the amount of virus progeny produced. This effect was reproduced in two very different cell types and with a selection of attenuated mutant viruses. Finally, several models were proposed that might account for this newfound effect of Rad51 on the Adenovirus life cycle. The data presented in this thesis shows that Adenovirus not only interacts with key molecular machinery within the host cell, but also manipulates this machinery to its own end. These data add additional layers of complexity to current knowledge of the virus/host cell relationship, and thus reveal new avenues of research for future work.

It is well known that the mitochondrial genome has a much higher spontaneous mutation rate than the nuclear genome. mtDNA mutations have been identified in association with many diseases and aging. mtDNA replication continues throughout the cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is required for replication and maintenance of the mitochondrial genome. However, it is not clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and nucleotide metabolism is associated with several human diseases. Clearly, to uncover the pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis, information is needed regarding dNTP biosynthesis and maintenance within mitochondria, and biochemical consequences of disordered mitochondrial dNTP metabolism. The studies described in this thesis provide important insight into these questions. First, we found that a distinctive form of ribonucleotide reductase is associated with mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP synthesis within mitochondria. Second, we found that long term thymidine treatment could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from thymidine treatment could account for the spectrum of mtDNA point mutations found in Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results support the proposed pathogenesis of this disease. Third, we found that normal intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity studies show that these imbalanced pools can stimulate base substitution and frameshift mutations, with a substitution pattern that correlates with mitochondrial substitution mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases. Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by insufficient transport of dNTPs into mitochondria resulting from a loss-of-function mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous mutant cells extracted from Dnc gene knockout mouse embryos. These results do not support the proposed pathogenesis of this disease and indicate that the DNC protein does not play a crucial role in the maintenance of intramitochondrial dNTP pools. / Graduation date: 2005

Graduation date: 1999

Graduation date: 1995

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 60-71).

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.