http://hdl.handle.net/1957/19991 Sun, 19 May 2013 09:10:39 GMT 2013-05-19T09:10:39Z http://hdl.handle.net/1957/37666 The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle Winzeler, Elizabeth A. The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle were investigated by studying the time-resolved fluorescence anisotropy decays of ethidium bromide intercalated into the DNA of the core particle. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. The anisotropy decays were collected using the method of time-resolved single-photon counting and were fit to a model developed by J. M. Schurr (Schurr, 1984) using a non-linear least squares fitting algorithm developed by the author for this purpose. It was shown that below a binding ratio of 0.1 there was no demonstrable change in the anisotropy as a function of binding ratio. Our results show, that the apparent torsional flexibility of DNA of to a nucleosome core particle is dependent on the number of base pairs of the DNA between points of attachment to the histone core. If this number is as high as 30 base pairs, then the torsional flexibility of DNA on a nucleosome core particle is as high or higher than DNA free in solution. Also, for reasonable values of N, the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the DNA on a core particle is highly constrained in its motions. The hydrogen ion concentration was shown to have a substantial effect on the fluorescent anisotropy decays, particularly in the early regions of the decay. These analyses indicated that the observed change could be attributed to either a loosening of the contacts between the DNA and the histone core, or a relaxing of the torsional flexibility of the DNA. Graduation date: 1991 Fri, 20 Jul 1990 00:00:00 GMT http://hdl.handle.net/1957/37666 1990-07-20T00:00:00Z http://hdl.handle.net/1957/37327 Towards the understanding of the function of the histone "tails" with respect to the structure, stability, and function of chromatin Dong, Feng By using immobilized trypsin, I have been able to prepare well-defined, stable trypsinized nucleosomes. The difficulties of lacking of control in the extent of trypsinization, which were encountered in previous studies with the use of free trypsin, have been eliminated. The nucleosomes and oligonucleosomes prepared by immobilized trypsin are suitable for biochemical and biophysical studies to analyze the function of the histone N-terminal regions ("tails"), which are removed by trypsin treatment, on chromatin structure and stability. Studies were first conducted using the trypsinized nucleosome core particles to examine the role of the histone tails in the stabilization of the nucleosome core particle. While it was found that these tails have little effect on either the nucleosome dissociation or the conformational transition in salt, they play a very important role in determining thermal stability of the nucleosome. The differential effects of selective removal of these tails also provided more insight about histone-DNA interactions in the nucleosome core particle. Experiments have also been carried out to investigate the change in structure and hydration of nucleosome core particles which may be associated with the salt-dependent conformational transition. Changes in the tertiary structures are suggested to be responsible for the salt-dependent transition. Roles of the histone tails in determination of nucleosome positions along specific DNA sequences were examined by analysis of nucleosome positioning on a specific eukaryotic gene sequence (Lytechinus Variegatus 5S rRNA gene) after in vitro nucleosome reconstitution with native and trypsinized histone octamers. Data obtained indicate that the histone tails are not required for nucleosome positioning. Results also seem to restrict the portions of histones which are responsible for determining nucleosome positions to the globular regions of (H3/H4)₂ tetramer, and possibly H2B. Studies with different DNA templates strongly suggest that the most important determinants of nucleosome positioning are the mechanical properties (such as bending and flexibility) of the DNA molecule. Taking together, it seems that the N-terminal tails of the histones may play roles in stabilizing both nucleosome structure and the higher-order structure of chromatin. Graduation date: 1991 Fri, 13 Jul 1990 00:00:00 GMT http://hdl.handle.net/1957/37327 1990-07-13T00:00:00Z http://hdl.handle.net/1957/37304 Large subunit of vaccinia cirus ribonucleotide reductase : affinity chromatography-based purification and photoaffinity labeling Warth, Rainer K. Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric regulation to the mouse enzyme and other eukaryotic reductases. The overall activity of the enzyme, which has two subunits (Rl and R2), is regulated through binding to ATP, which activates the enzyme, and dATP which seryes as an inhibitor. Both nucleotides bind to the same allosteric site, called the activity site, on the large subunit of RR. The specificity of the enzyme towards the four ribonucleoside diphosphate substrates is regulated by the binding of ATP, dATP, dTTP and GTp. Each of these nucleotides affects the reduction of a specific nucleoside diphosphate. Although this enzyme's allosteric regulation is kinetically well understood it has not been possible so far to gain further structural information about the location of the activity site and specificity site. The use of deletion mutants and photoaffinity labeling of the large subunit to identify the location of the binding sites is the incentive for this thesis. With the introduction of 6xHis/Nickel Nitrilo-tri-acetic acid (Ni-NTA) chromatography, the purification of the large subunit was improved in the E. coli and vaccinia virus/T7 RNA polymerase hybrid system. The purification of several deletion mutant forms of the large subunit was also attempted, but it was not possible to purify any of them from either of the expression systems. The purified full-length large subunit obtained with the Ni-NTA-chromatography system was used for a photoaffinity labeling experiment with [³²P]dATP and [³²P]dTTP. The labeled proteins were proteolytically digested to find out about the specificity of the labeling experiment and also to map the binding site of the nucleotide. It was found that labeling of dATP yielded few discrete bands indicating specific binding, while a comparable experiment with dTTP indicated less specific binding, based on a larger number of labeled bands. In competition experiments with non-radioactive nucleotides, vaccinia virus R1 featured the same properties as the mouse and E. coli counterparts. This is consistent with data from kinetic experiments, which also establish the same kinetic properties between vaccinia virus RR with those of mouse and E. coli (RR). To identify the sequence of the fragments carrying the label the digests were subjected to mass spectrometric analysis. However, it was not possible to determine the sequence of the labeled fragment by mass spectrometry due to poor spectral resolution. Graduation date: 1994 Tue, 31 Aug 1993 00:00:00 GMT http://hdl.handle.net/1957/37304 1993-08-31T00:00:00Z http://hdl.handle.net/1957/37268 Enzyme associations in deoxyribonucleotide biosynthesis : anti-idiotypic antibodies as probes for direct protein-protein interactions Young, James Patrick The ability to faithfully replicate DNA is dependent upon the maintenance and regulation of its precursors, the deoxyribonucleoside triphosphates. Enzymes encoded by the bacteriophage T4 have been widely used as models of biochemical processes. A body of evidence supports the concept that the bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are associated as a complex within the infected Escherichia coli. This dissertation describes the continued examination of the protein-protein interactions involved in deoxynucleotide biosynthesis of bacteriophage T4. My studies on the protein-protein interactions involved in deoxyribonucleotide biosynthesis focused on two unique phage proteins, the dCMP hydroxymethylase enzyme and the translational regulator RegA. An initial study was undertaken to determine if the generation of anti-idiotypic antibodies would prove useful in the identification of direct interactions between dCMP hydroxymethylase and other proteins of the deoxyribonucleotide synthetase complex. For the initial generation of anti-idiotypic antibodies, polyclonal rabbit antibodies were generated to affinity purified anti-dCMP hydroxymethylase polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the bacteriophage T4 dTMP synthase. A second method to generate anti-idiotypic antibodies was attempted with the translational regulator RegA. The generation of anti-idiotypic antibodies to the RegA protein involved the purification of anti-RegA rabbit Fab fragments and the generation of anti-anti-RegA polyclonal antibodies within mice. This alternative method was determined to be inferior to the initial method for generating anti-idiotypic antibodies. Additional studies involved the examination of RegA protein-protein interactions using affinity chromatography. A number of bacteriophage T4 early proteins were determined to associate with an immobilized RegA column. Graduation date: 1992 Mon, 11 May 1992 00:00:00 GMT http://hdl.handle.net/1957/37268 1992-05-11T00:00:00Z