The retinoblastoma tumor suppressor protein (RB) is an important regulator of the cell cycle and development. Significantly, RB is inactivated in a majority of human cancers. Thus, elucidating the function of RB will give us a better understanding of how it prevents cancer. Many decades of research have yielded a detailed understanding of the role of RB in cell proliferation through transcriptional repression of target genes. However, the precise mechanisms of its action in many cellular pathways are poorly understood, including the control of DNA replication and post-transcriptional control of gene expression. Drosophila melanogaster presents a simplified genetic system to study cancer genes. Several published observations have suggested a role for RB in regulating DNA replication. Interestingly, other data indicate that RB associates with RNA processing factors. I have characterized novel protein-protein interactions with the Drosophila retinoblastoma tumor suppressor homologue Rbf, with an emphasis on its poorly characterized N-terminal domain. I describe the interaction of Rbf with the origin recognition complex, indicating a unique connection to DNA replication control. I also show that Rbf interacts with the RNA binding protein Squid, and review the literature that suggests potential role of RB/E2F in the control of RNA processing. The ability to control RNA processing may be an additional, unappreciated mode of gene regulation by RB. A focused study of the uncharacterized amino-terminal domain of Rbf has revealed new details about the retinoblastoma tumor suppressor in cell regulation, including DNA replication and RNA processing.

科学研究費補助金 研究種目:基盤研究(C) 課題番号:16570143 研究代表者:小川 徹 研究期間:2004-2005年度

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed. In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

The essential cell cycle kinase Dbf4/Cdc7 (DDK) triggers DNA replication through phosphorylation of the Mcm2-7 helicase at replication origins. Prior work has implicated various Mcm2-7 subunits as targets of DDK, however it is not well understood which specific subunits mediate the docking of the DDK complex. Through yeast two-hybrid and co-immunoprecipitation analyses, we found that Dbf4 and Cdc7 interact with distinct subunits of the Mcm2-7 helicase complex. Dbf4 showed the strongest interaction with Mcm2 while Cdc7 associated with Mcm4 and Mcm5. Dissection of the N-terminal region of Mcm2 revealed two regions that mediate the interaction with Dbf4, whereas in Mcm4, a region near the N-terminus has been previously identified by another group as the DDK docking domain. Mutant forms of Mcm2 (Mcm2ΔDDD) or Mcm4 (Mcm4ΔDDD) lacking the DDK docking domain were expressed in cells and resulted in modest growth and replication defects. Combining the two mutations resulted in synthetic lethality, suggesting a redundant mechanism of Mcm2 and Mcm4 in targeting the DDK complex to Mcm rings. Furthermore, growth inhibition could be induced in a Mcm4ΔDDD background by overexpressing Mcm2 to titrate Dbf4 from Mcm rings. These growth defects were exacerbated in the presence of genotoxic agents such as hydroxyurea and methyl methanesulfonate, suggesting that DDK-Mcm interactions may play a role in stabilizing replication forks under S-phase checkpoint conditions. Regions of Cdc7 were examined for their interaction with Mcm4 and Dbf4. Results have shown that the N-terminal amino acid region 55-124 and the C-terminal region 453-507 of Cdc7 are likely target regions for Dbf4-binding. Several conserved residues were identified within the N-terminal 55-124 Cdc7 region that interface with conserved residues within motif-C of Dbf4. Conserved residues were identified within the DDD domain of Mcm2 and mutating these residues resulted in a decreased interaction with Dbf4. Lastly, bioinformatics analysis has revealed potential conserved residues within the Mcm4DDD region, which may play a role in binding to Cdc7. This research is significant because these factors, which are conserved in all eukaryotes studied to date, should give further insight as to how DNA replication is triggered and how it is affected when cells are exposed to DNA damaging or replication compromising agents. This research also has implications in cancer genetics, as prior studies have shown elevated DDK and Mcm protein levels in tumour cell lines and melanomas, with Cdc7 showing great promise as a cancer therapeutic target. Such knowledge will further enhance our understanding of the DNA replication process and the roles of cell cycle proteins involved, under both normal and checkpoint conditions.

The expansion of gene-specific trinucleotide repeats is responsible for a growing list of human disorders, including myotonic dystrophy type 1 (DM1). Repeat instability for most of these disorders, including DM1, is characterized by complex patterns of inherited and ongoing tissue-specific instability and pathogenesis. While the mechanistic basis behind the unique locus-specific instability of trinucleotide repeats is currently unknown, DNA metabolic processes are likely to play a role. My thesis involves investigating the contribution of DNA replication to the trinucleotide instability of myotonic dystrophy type 1. Herein I have designed an in vivo primate model system, based on the SV40 replication system, to assess the contribution of DNA replication to DM1 repeat instability. This system allows the assessment, under controlled conditions, and manipulation of variables that may affect replication-associated repeat instability, under a primate cellular system. Using the SV40 model system, I not only confirmed previous observations that repeat length and replication direction affect repeat instability, but also for the first time determined that the location of the replication origin relative to the repeat tract plays an important role in repeat instability. This novel observation allowed for the development of a fork-shift model of repeat instability, in which cis-elements adjacent to the repeat tract affect replication, in turn altering the propensity for repeat instability. To further my study of DNA replication in DM1 repeat instability, I have mapped the origin of replication adjacent to the DM1 locus in human patient cells and the tissues of DM1 transgenic mice actively undergoing repeat instability. The position of the replication origins adjacent to the repeat tract at the DM1 locus places several known cis-elements, including CTCF binding sites, in a position to alter replication as predicted by the fork-shift model. My analysis of the CTCF sites showed them capable of altering replication and repeat instability at the DM1 locus. Taken together these results suggest that the placement of replication origins, repeat tracts and cis-elements, may mark trinucleotide repeat tracts, such as the DM1, for locus-, tissue- and development-specific replication-associated repeat instability.

Errata sheet pasted opposite Table of Contents. / Includes bibliographies. / 142, [171] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the UV-induced inhibition of oriC initiation function and the nature of the phenotype of the mutant origin of replication of Escherichia coli, oriCc. The specific aims of this study are to delineate the source of the UV-induced trans-acting inhibition of oriC function, utilizing the phage vector [lambda]poriCc, and to demonstrate the oriCc allele as an enhanced DNA replication initiator from oriC. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997?

Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 15, 2008). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.