The Saccharomyces cerevisiae plasmid, 2 micron circle, resides in the yeast nucleus at a high copy number. It provides no apparent growth advantage to its host, nor imposes any significant growth disadvantage. The plasmid is an excellent paradigm for studying mechanisms utilized in the persistence of a eukaryotic selfish DNA element that is selectively neutral. The plasmid achieves stable propagation and copy number maintenance by combining a partitioning system and an amplification system. The partitioning proteins Rep1p and Rep2p promote the recruitment of the histone H3 variant Cse4p and the yeast cohesin complex to the partitioning locus STB during S phase, leading to the formation of a functional partitioning complex which segregates the plasmid equally during mitosis. The integrity of the mitotic spindle is a pre-requisite for the specific nuclear localization of the plasmid as well as for plasmid association with a subset of the partitioning proteins such as Cse4p and the cohesin complex. The work presented in this thesis reveals, using tools of molecular genetics and cell biology, the involvement and possible functions of a microtubule associated nuclear motor protein, Kip1p, in the 2 micron circle partitioning pathway. The plasmid missegregates in kip1[Delta] cells, but not in cells harboring deletions of genes coding for the other nuclear motors. Kip1p interacts with the plasmid partitioning system and promotes the association of Cse4p and the cohesin complex with STB. Lack of Kip1p function delocalizes the plasmid from its characteristic nuclear locale in close proximity to the spindle pole body. The distance between a reporter plasmid and the spindle pole body is nearly doubled in a kip1[Delta] host strain. We propose that, unlike the conventional roles played by nuclear motors in spindle function and chromosome segregation, the Kip1p motor assists the 2 micron circle in associating with the mitotic spindle and translocating to its ‘partitioning center’. / text

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that catalyzes the cleavage of NAD⁺ into nicotinamide and ADP-ribose moiety, the latter of which may be covalently attached as a branched polymer of poly(ADP-ribose) to PARP-1 itself (automodification) or to other nuclear acceptor proteins (transmodification). PARP-1 plays pivotal roles in many fundamental biological processes, including DNA repair, gene expression, cell death and cell cycle regulation. The multiple functions of PARP-1 in various cellular events correlate well to its roles in carcinogenesis, inflammatory response, neural function, and aging. PARP-1 has a modular organization comprising six independent domains (domain A-F). Each domain has its own characteristic function in PARP-1 enzymatic catalysis. In this dissertation, the solution structure of domain C was determined by multi-dimensional NMR spectroscopy. To complement the structural results, the requirement of domain C for PARP-1 catalysis was demonstrated using activity assays. This structure-function relationship study will help to unveil the mechanism of the PARP-1 reaction, and should provide valuable information for the design of more potent and selective PARP-1 inhibitors. The determination of poly(ADP-ribosyl)ation sites is critical for understanding the biological roles of this modification. However, the identification of poly(ADPribosyl)ation sites has countered some daunting technical limitations due to the difficulties resulting from the heterogenous nature of this modification. In this dissertation, a methodology based on mass spectrometry is developed and used to identify ADP-ribosylation sites within the automodification domain (domain D) of PARP-1. Using this method, we were able to unambiguously localize three ADPribosylation sites on domain D. This method can be readily applied to study the transmodification of other substrates as well as PARP-1 automodification. As many as seventeen PARP homologues exist in the human proteome. The functional redundancy of the multiple PARP proteins has complicated the analysis of mammalian PARP-1 function in vivo. We have probed the biological roles of PARP-1 using an artificial PARP-1 pathway in yeast, an organism lacking the endogenous PARP-1. Our data suggest the heterologously expressed human PARP-1 in yeast retains some similar functions as it does in mammalian cells. Furthermore, a new function of PARP-1 in ribosome biogenesis was proposed. / text

Although influenza is usually acute self-limiting respiratory infection, influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the problem is that the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverage are urgently needed to combat with the disease. Since nucleoprotein (NP), which is encoded by influenza virus genome, is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle, numerous studies are focused on this protein in attempts to develop broad-spectrum anti-influenza therapeutics. Recently, Kao et al. found that the addition of a novel small molecule nucleozin could lead to large aggregates of NP, which in turn caused cessation of virus replication. Give that the interaction between NP and nucleozin is still not unveiled, it is crucial to identify the binding sites using X-ray crystallography. The full length influenza A/WSN/33 (H1N1) NP gene was cloned into pET28 vector, with His-tag in its C-terminus and overexpressed in E.coli strain Rosetta 2. Cell culture was purified by HisTrap HP and Superdex-200 16/60 gel filtration columns. Crystals were grown using the vapour diffusion method and the NP-nucleozin complex was prepared by soaking native crystal in solution containing 0.25 mM nucleozin for 2h. Crystals of the complex can diffract to 3.0 Å at the Shanghai Synchrotron Radiation Facility. The structure of NP was determined by molecular replacement and it belongs to space group C121 with two NP trimers per asymmetric unit. After further refinement, two nucleozin molecules were found in each asymmetric unit, and each of them could bind with two NP molecules at the same time. The ligand binding pockets were formed by the combination of Y289/N309 pocket from one NP molecule, and R382 pocket from another NP molecule. Therefore, the function of nucleozin is to bridge two NP molecules and lead to NP aggregation, which are in agreement with functional studies on nucleozin. Furthermore, computational models of the NP-nucleozin binding are provided to reveal the mechanism of nucleozin induced aggregation. In addition, recent work on interaction between NP and another novel molecule named compound A has also been briefly described and compared with NP-nucleozin complex at the end of this thesis. Collectively, this study presents a new paradigm for better understanding of how NP and nucleozin interact with each other and hence result in NP aggregates, which is envisaged to accelerate the development of anti-influenza therapeutic agents. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Not available / text

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Harvey, Stephen C; Committee Member: Hud, Nicholas V; Committee Member: McCarty, Nael A; Committee Member: Wartell, Roger M.

Thesis (Ph. D.)--Michigan State University. Dept. of Microbiology and Molecular Genetics, 2006. / Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 114-125) Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Andrew D. Ellington. Vita. Includes bibliographical references. Available also from UMI company.