Der Replikation des NV-Genoms geht die kotranslationale Spaltung des vom ORF1 kodierten Polyproteins durch die viruseigene Protease, voraus. Erst in cis und in trans Prozessierungen an definierten Spaltmotiven innerhalb des Polyproteins ermöglichten die Freisetzung der strukturellen und nicht-strukturellen Proteine, die für den Aufbau des Replikationskomplexes und für die Initiation der Replikation essentiell sind. Die Regulation der Polyproteinprozessierung war allerdings bislang unbekannt. In der Charakterisierung der sequentiellen und differentiellen Prozessierung des Polyproteins bestand demnach die Hauptaufgabe dieser Arbeit. Dafür wurde zunächst ein NV-Volle-Länge-cDNA-Klon aus sechs sich überlappenden cDNA-Einzelfragmenten, unter Verwendung der overlap-extension PCR, hergestellt. Dieser Volle-Länge cDNA-Klon diente als Template für die Generierung von Vorläuferkonstrukten, die für die Charakterisierung der in cis und in trans Prozessierung des Polyproteins verwendet wurden. Die cis-Spaltung des Polyproteins konnte sowohl in E.coli als auch in humanen 293-T Zellen anhand eines Vorläuferproteins, das die komplette Sequenz der 3CLpro enthält, die 5`-seitig von der Spaltstelle zum VPg Protein und 3`-seitig vom N-Termins der 3DLpol flankiert ist, verifiziert werden. Infolge der kotranslationalen cis-Spaltung dieses Vorläuferproteins kam es zur Freisetzung der 3CLpro, die anschließend aufgereinigt wurde. Zusätzlich wurde das Fusionsprotein 3CLproµE1189A3DLpol aufgereinigt, bei dem das Spaltmotiv E/G zwischen der 3CLpro und der 3DLpol so mutiert wurde, dass die kotranslationale Spaltung des Fusionsproteins verhindert wurde. Anhand der 3CLpro sowie dessen Vorläufers 3CLproµE1189A3DLpol sollte die differentielle und sequentielle Spaltung des Polyproteins in trans charakterisiert werden. Dafür wurden synthetisch hergestellte Peptide, die die authentischen Spaltmotive innerhalb des Polyproteins aufwiesen, sowohl mit der 3CLpro als auch mit der 3CLproµE1189A3DLpol in einem trans-Spaltungsassay inkubiert und anschließend die Spaltung mit Hilfe der reversed-phase Chromatographie analysiert. Im Rahmen dieser Versuche konnte gezeigt werden, dass nur die Spaltmotive zwischen dem N-terminalem Protein p37 und der 2CNTPase sowie zwischen der 2CNTPase und dem Protein p20 in trans von der 3CLpro gespalten wurden. Massenspektrometrische Analysen wiesen nach, dass die Spaltungen jeweils zwischen den Aminosäureresten Q/G stattgefunden hatte. Zusätzlich zu den Peptiden p37/2CNTPase und 2CNTPase/p20 konnte das Peptid VPg/3CLpro durch das Fusionsprotein 3CLproµE1189A3DLpol an der Schnittstelle E/G prozessiert werden. Anhand von Berechnungen zur relativen Spalteffizienz von 3CLpro und 3CLproµE1189A3DLpol wurde nachgewiesen, dass 3CLpro eine signifikant höhere Spezifität zu den Spaltmotiven der Peptide p37/2CNTPase und 2CNTPase/p20 aufwies als das Fusionsprotein 3CLproµE1189A3DLpol. Die Spaltspezifität der 3CLpro war dabei an dem Spaltmotiv zwischen der 2CNTPase und dem Protein p20 signifikant höher als an dem Spaltmotiv zwischen dem N-terminalem Protein p37 und der 2CNTPase. In vitro Translationsstudien des NV-ORF1 im zellfreien System bestätigten, dass die kotranslationale in trans-Spaltung des Polyproteins nur zwischen dem Protein p37 und der 2CNTPase und der 2CNTPase und dem Protein p20 stattfand. Darüber hinaus konnte anhand dieses Versuches gezeigt werden, dass die initiale Prozessierung des Polyproteins auf der trans-Aktivität der 3CLpro beruht, was in der Freisetzung der Proteine p37, 2CNTPase und des Fusionsproteins VPg3CLpro[delta]3DLpol resultiert. Eine weitere Spaltung von VPg3CLpro[delta]3DLpol konnte im Rahmen dieses Versuches nicht gezeigt werden. Mit Hilfe des humanen Zellkultursystems sollte anschließend untersucht werden, ob zelluläre Faktoren in die Prozessierung des p20VPg Vorläuferproteins involviert sind. Dafür wurden Koexpressionsstudien des Vorläuferproteins p20VPg mit dem 3CLpro Vorläuferprotein [delta]VPg3CLpro[delta]3DLpol durchgeführt. Doch auch in dieser Expressionsstudie konnte eine Spaltung des Vorläuferproteins nicht beobachtet werden. Interessanterweise wurde allerdings die cis-Spaltung des Vorläuferproteins [delta]VPg3CLpro[delta]3DLpol durch die Koexpression mit [delta]VPg3CLpro[delta]3DLpol verhindert. Unter Verwendung des trans-Spaltungsassays konnte daraufhin in vitro verifiziert werden, dass p20VPg die Aktivität der 3CLpro am Peptid p37/2CNTPase signifikant reduzierte. Der initiale Schritt der NV-Replikation basiert auf der Aktivität der 3CLpro. Eine Inaktivierung dieses Enzyms würde demnach den Replikationszyklus verhindern. Somit stellt dieses Enzym ein geeignetes Target für anti-NV Medikamente dar. Aus diesem Grund wurden in dieser Arbeit mögliche 3CLpro Inhibitoren getestet. Anhand dieser Testreihen konnte gezeigt werden, dass die Substanz CMK eine mögliche Grundlage, für die Entwicklung von NV-3CLpro spezifischen Chlormethylketon-Peptidanaloga als Inhibitoren, darstellen könnte.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 168-176). Also available on the Internet.

Thesis (Ph. D.)--University of Toledo, 2008. / Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry." Includes bibliographical references (leaves 305-309).

Thesis (Ph. D.)--University of Chicago, Department of Molecular Genetics and Cell Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 168-176). Also available on the Internet.

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G1, is suppressed by Geminin, and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S-phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading, and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G1 relative to S-phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G1 and repressed by Geminin-HDAC11 association with Cdt1 in S-phase, and represents a novel form of replication licensing control.

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. Two components of this coordination are the SOS system and nucleoid occlusion. The SOS regulon expresses DNA repair genes after DNA damage and delays FtsZ-ring formation and cell division to enhance survival. Nucleoid occlusion prevents cell division over un-replicated nucleoids, a process partially dependent on the SlmA protein. Z-ring formation is shown here to be dependent on DNA replication by an additional mechanism, independent of the SOS regulon and of the SlmA protein and which acts by preventing FtsZ-ring formation when replication is perturbed. Replication dependent Z-ring formation (RDZ) was shown to be SOS-independent by the fact that FtsZ-rings were inhibited, after replication blockage, in a lexA1 mutant and in strains containing a null allele of sulA or the ftsZ/sulB103 mutation. SlmA protein-independence was shown by the fact that FtsZ-rings were also inhibited in lexA1 [Delta]slmA double mutants after replication blockage. This SOS- and SlmA-independent mechanism functions effectively in cells growing slowly with only one replicating chromosome and also in cells growing rapidly with multi-fork replication and after replication inhibition by different methods - chemical inhibitors and a temperature-sensitive polymerization mutation. / text

Mitochondrion is an organelle found in the eukaryotic cell. It is responsible for essential metabolic processes as well as ATP production via oxidative phosphorylation (OXPHOS). The mitochondrion contains DNA that encodes for several subunits in the OXPHOS system as well as rRNA and tRNA for translation. It also has its own replication, transcription and translation machinery. Proper maintenance of the mitochondrial DNA is critical for the cell’s health. Saccharomyces cerevisiae mitochondrial transcription system has been a great model system for its ease of genetic manipulation as well as having conserved RNA polymerases across species. The polymerases are homologues to T7 RNA polymerase, but have longer N-terminal domain and require transcription factor(s). The reason for the extra domain as well as the need for an accessory factor is still unclear. This study reveals the role of Rpo41 N-terminal domain (NTD) as well as clarifies the role of Mtf1, the transcription factor, in transcription initiation. Rpo41 is the 153 kDa catalytic subunit, and Mtf1 is 40 kDa, the transcription factor of the yeast mitochondria. We have shown that Mtf1 is required for correct promoter sequence recognition as well as inhibition of incorrect initiation. Although it was thought that Rpo41 has intrinsic promoter recognition capability, we have shown that Rpo41 can initiate transcription on a pre-melted DNA, even if it is not the consensus promoter sequence. N-terminal truncation mutant studies showed that the NTD of Rpo41 is also required for correct transcription initiation. On linear duplex DNA, N-terminal truncation of 321 amino acids has little effect when Mtf1 is present. On pre-melted DNA, it shows opposite trend from the wild-type. 160 N-terminal amino acid residue truncation shows little activity, whereas Mtf1 increases activity, even on non-promoter initiation sites. We further investigated properties of Rpo41 in replication. A link between mitochondrial transcription and replication has been suggested before, where Rpo41 functions as the leading strand primase. Our studies show that Rpo41 can indeed function as the leading and lagging strand primase, and explains why Rpo41 is able to initiate transcription on non-promoter sites. N-terminal truncation resulted in loss of primase activity, which shows that NTD is required for replication. / text

While it is now understood that long-standing hypertension is predictive of later cognitive decline and risk for dementia, little research attention to date has focused on whether the short-term dynamics of blood pressure exert immediate influence on cognitive functioning. The present study contributes to this growing field with a conceptual replication and extension of work by Gamaldo, Weatherbee and Allaire (2008). A sample of 27 older adults (M=70.2 years) completed daily assessments of blood pressure, psychological stress and cognitive functioning for 14 consecutive days. Multilevel models conditional on demographic factors were applied to simultaneously estimate between- and within-person effects across three metrics of blood pressure (systolic, diastolic and pulse pressure) and five measures of cognitive functioning. To follow a suggestion proposed by Gamaldo et al., the model was extended to include main effect and blood pressure interaction terms for stress at both levels. In secondary analyses, within-person mediation models were applied to explore blood pressure as a mediator between stress and cognition. Results from the first model demonstrated a direct, positive association between occasion diastolic pressure and episodic memory. A cross-level interaction term revealed that processing speed was impaired on high-diastolic pressure days for those with high diastolic pressure on average. We found no evidence that occasion blood pressure mediated the association between stress and cognition. Overall, our results align with the hypothesis that age-related changes to vascular structures impair the carrying capacity of blood vessels and that occasions of increased blood pressure provide additional force to overcome these limitations, delivering larger quantities of blood and oxygen to cerebral tissue. We conclude that upward fluctuations in diastolic pressure may be cognitively beneficial for older adults; diastolic pressure is the most sensitive metric for detection of within-person associations with cognition; and episodic memory and processing speed exhibit sensitivity to occasion blood pressure levels. / Graduate

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.