Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo.

In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.

Circular dichroism (CD) spectra were obtained for bacteriophage T4 lysozyme and three of its mutants in the presence and absence of colloidal silica nanoparticles. Mutant lysozymes were produced by substitution of the isoleucine at position 3 with tryptophan, cysteine and leucine. Each substitution resulted in an altered structural stability, quantified by a difference in free energy of unfolding from the wild type. CD spectra recorded in the absence of colloidal silica agreed with x-ray diffraction data in that the mutants and wild type showed similar secondary structures. CD spectra of protein-nanoparticle complexes recorded after contact for 90 minutes showed significant differences from those recorded in the absence of nanoparticles, and these differences varied among the proteins. The percentage of a-helix lost in these proteins upon adsorption to silica nanoparticles was also recorded as a function of time by CD. For a 1:2 protein to particle mixture, different kinetic behaviors were observed among the proteins. The more unstable the protein, the greater the rate and extent of secondary structure loss upon adsorption. For a 1:1 protein to particle mixture, only results recorded with the tryptophan mutant were significantly different from the other variants. The kinetic data recorded for the 1:2 protein to particle ratio was evaluated using two different protein adsorption models. Both models allow proteins at an interface to exist in two different states: state 1 molecules retain their native conformation, while state 2 molecules lose a certain amount of their native secondary structure and occupy more surface area than state 1 molecules. The main difference between these two models is that one allows state 2 molecules to be adsorbed directly from solution, while the other requires that state 2 molecules be generated by surface-induced conversion of state 1 molecules. The former model showed a better fit to the data than the latter from a least squares comparison. Both models indicated that proteins of lower thermal stability have a greater tendency to adopt state 2 on silica. / Graduation date: 1997

Graduation date: 1995

Graduation date: 1993

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

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).

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

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 160-180). Also available on the World Wide Web.

Silicification of organisms in silica-depositing environments can impact both their ecology and their presence in the fossil record. Although microbes have been silicified under laboratory and environmental conditions, viruses had not been, prior to this work. Bacteriophage T4 was successfully silicified under laboratory conditions that closely simulated those found in silica-depositing hot springs. Virus morphology was maintained during the short period of silicification (48 hours), and a clear elemental signature of silicon and phosphorus was detected by energy-dispersive X-ray spectrophotometry (EDX). However, the EDX signature of silicified virus was not sufficiently distinct from that of cell membrane or phosphate minerals for that technique to be used to discover viral remains in hot spring mineral deposits. Having shown that bacteriophage T4 can be silicified, it was then determined that the impact of silica exposure on infectivity varied widely between different viruses. The effect on infectivity did not appear to be related to virus size or morphology. In addition, the impact on infectivity was at least partially reversible, indicating that it was caused, at least in part, by occluding infection-related structures on the virus, rather than destruction or denaturation of the virus. Those viruses which showed a decline in infectivity with silica exposure also showed increased resistance to desiccation after being exposed to silica, which has implication for long-range virus dispersal. The desiccation resistance was proportional to the degree that silicification reduced infectivity in that virus. Desiccation resistance also declined with prolonged exposure to drying, suggesting that the mechanism was due to the silica coating helping to retain water rather than replacing the hydrogen bonding of water. Virus dispersal is critical for both the spread of disease and the diverse roles that viruses play in Earth ecology. However, the mechanisms of host-independent virus dispersal are poorly understood and hotly debated. These experiments showed that, under mild conditions, diverse viruses can be coated in silica and that silica coating provides some, if not most, viruses with remarkable desiccation tolerance. Virus silicification thus provides a potential mechanism for global dispersal of viruses that could not otherwise tolerate the desiccation of wind-borne transportation.