Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 162-178). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 101-112).

No description available.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Initiation of DNA replication. Department of Biochemistry and Molecular Genetics. Includes bibliographical references (142-171). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Mammalian DNA replication initiation. Includes bibliographical references (219-245). Also available online through Digital Dissertations.

Tetrahydrofolate-mediated one-carbon metabolism is required for the biosynthesis of many central metabolites, including some amino acids, nucleobases, and nucleotides, and hence dysfunction of one-carbon metabolism is associated with many human diseases and disorders. The mitochondrial glycine decarboxylase complex (GDC) is an important component of one-carbon metabolism, generating 5,10-methylene-tetrahydrofolate (5,10-CH2-H??4folate) from glycine. Previous work has shown that the genes encoding the unique sub-units of the Saccharomyces cerevisiae GDC (GCV1, GCV2 and GCV3) are regulated in response to changes in the levels of cytosolic 5,10-CH2-H??4folate (Piper et al., 2000). Given the centrality of 5,10-CH2-H??4folate to many aspects of metabolism, it was hypothesised that other genes may be regulated by the same mechanism. Using microarray analysis of S. cerevisiae under a number of conditions that affect 5,10-CH2-H??4folate levels, the ??one-carbon regulon??, a group of genes that were co-regulated with the GCV genes was identified. The one-carbon regulon corresponds closely to genes whose promoters are bound by the purine biosynthesis regulator Bas1p, but not all one-carbon regulon members are significantly purine regulated. Genetic approaches demonstrated that the one-carbon unit response and the purine response are distinct, though both depend on the presence of Bas1p. This demonstrated that the close metabolic connections of one-carbon and purine metabolism are reflected in over-lapping, but separable regulatory mechanisms. The identity of the sensor of one-carbon unit depletion remains unknown, but in the course of investigation of the candidate regulator Caf17p, it was demonstrated that Caf17p is in fact involved in Fe/S cluster protein maturation. Examination of the effects of Caf17p depletion revealed that Caf17p is required for the function and maturation of the related mitochondrial Fe/S proteins aconitase and homoaconitase, as well as the function of, but not de novo iron incorporation into, the mitochondrial radical-SAM Fe/S protein biotin synthase. Because other Fe/S proteins were unaffected, Caf17p appears to be a specialised Fe/S maturation factor. The presence of a putative H4folate binding site indicates that Caf17p may constitute a metabolic link between one-carbon and iron metabolism.

Tetrahydrofolate-mediated one-carbon metabolism is required for the biosynthesis of many central metabolites, including some amino acids, nucleobases, and nucleotides, and hence dysfunction of one-carbon metabolism is associated with many human diseases and disorders. The mitochondrial glycine decarboxylase complex (GDC) is an important component of one-carbon metabolism, generating 5,10-methylene-tetrahydrofolate (5,10-CH2-H??4folate) from glycine. Previous work has shown that the genes encoding the unique sub-units of the Saccharomyces cerevisiae GDC (GCV1, GCV2 and GCV3) are regulated in response to changes in the levels of cytosolic 5,10-CH2-H??4folate (Piper et al., 2000). Given the centrality of 5,10-CH2-H??4folate to many aspects of metabolism, it was hypothesised that other genes may be regulated by the same mechanism. Using microarray analysis of S. cerevisiae under a number of conditions that affect 5,10-CH2-H??4folate levels, the ??one-carbon regulon??, a group of genes that were co-regulated with the GCV genes was identified. The one-carbon regulon corresponds closely to genes whose promoters are bound by the purine biosynthesis regulator Bas1p, but not all one-carbon regulon members are significantly purine regulated. Genetic approaches demonstrated that the one-carbon unit response and the purine response are distinct, though both depend on the presence of Bas1p. This demonstrated that the close metabolic connections of one-carbon and purine metabolism are reflected in over-lapping, but separable regulatory mechanisms. The identity of the sensor of one-carbon unit depletion remains unknown, but in the course of investigation of the candidate regulator Caf17p, it was demonstrated that Caf17p is in fact involved in Fe/S cluster protein maturation. Examination of the effects of Caf17p depletion revealed that Caf17p is required for the function and maturation of the related mitochondrial Fe/S proteins aconitase and homoaconitase, as well as the function of, but not de novo iron incorporation into, the mitochondrial radical-SAM Fe/S protein biotin synthase. Because other Fe/S proteins were unaffected, Caf17p appears to be a specialised Fe/S maturation factor. The presence of a putative H4folate binding site indicates that Caf17p may constitute a metabolic link between one-carbon and iron metabolism.

Thesis (M.Sc. (Biochemistry)--Mahidol University, 1982.

Solid-state NMR measurement at cryogenic temperature shows significant potential for biological analysis due to its advantages for sample stability and detection sensitivity. However, at low temperature lineshape broadening and low spectral resolution are commonly observed and limit the applications for complex protein systems. Here, we explored the hypotheses for the underlying mechanisms of broad linewidths at low temperatures by studying E. coli Dihydrofolate reductase (DHFR). Our results support the hypothesis that conformational heterogeneity is a major source of linebroadening. We measured the protein backbone torsion angle (Ψ) at 105 K. In a selectively enriched protein sample with only one amide 13 C’- 15 N correlation expected, we identified three different conformations with distinct N chemical shift values accounting for the dramatic broadening observed in low temperature NMR spectra. We presume that backbone torsion angle fluctuates among the conformers on picosecond timescale at room temperature and are ‘frozen out’ giving rise to static heterogeneity at cryogenic temperatures. MD simulations support this hypothesis. QM/MM predicted chemical shifts based on snapshots from a MD simulation show excellent agreement with our data in that the average agrees well with the room temperature shift and the distribution agrees well with the low temperature spectral lineshape. On the other hand, our data suggest that there is no relationship between the μs - ms motions at room temperature and the lineshape broadening at low temperature. Resonance assignments of the apoenzyme in solution and associated liganded states were accomplished to identify the conformational transition in chemical exchange. We analyzed the 15 N relaxation dispersion profile of each residue at room temperature in solution; the rates appear to be organized in functional groups that exchange in a concerted fashion, with shift differences related to ligated-vs-unligated changes. The chemical shift changes associated with μs - ms exchange phenomena (and with ligation) are about an order of magnitude too small to explain the low temperature lineshapes, and also have no correlation with the low temperature lineshapes.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 92-99).