A new approach in mapping restriction fragments by means of primed extension was proposed but was found to be unfeasible after studying the extent of T4 polymerase mediated DNA synthesis. The maximum length of DNA replication mediated by T4 polymerase was studied using ØX-174 DNA as template primed by a restriction fragment of the same DNA. Both nucleotide incorporation kinetics and alkaline gel electrophoresis were used to study the products of DNA synthesis. Although the incorporation kinetics suggested that the primer was extended by approximately 100 nucleotides, the electrophoretic mobilities of the products suggested much less extension. The effect of T4 gene 32 protein (unwinding protein) was also studied. This protein was purified by DNA cellulose chromatography to near homogeneity and was shown to be nuclease free. The purified protein stimulated nucleotide incorporation three-fold when added to the usual T4 polymerase reaction mixture. Contrary to the kinetic results, however, the gel mobilities of the products again showed only limited extension of the primer. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Experiments with ØW-14-infected, thymidine-requiring mutants of P_. acidovorans strain 29 demonstrated that deoxyuridine but not thymidine was a precursor of thymine in ØW-14 DNA. Deoxyuridine was also a precursor of the a-putrescinylthymine found in ØW-14 DNA. The biosynthesis of a-putrescinylthymine and thymine was mediated by enzyme activities appearing after infection. ØW-14 DNA synthesis and DNA modification was resistant to the antibiotics trimethoprim and 5-fluorodeoxyuridine. This indicated that endogenous thymidine biosynthesis was unlike that observed in the uninfected host or in other biological systems. These observations helped demonstrate that hydroxy-methyluracil-containing nucleotides were precursors of thymine and a-putrescinylthymine-containing nucleotides (Neuhard et al., 1980). The absence of a-putrescinyl thymine and thymine nucleotides in 0W-14-infected cell nucleotide pools suggested that these nucleotides might be synthesized from hydroxymethyluracil at the polynucleotide level. Degradative analysis of nascent ØW-14 DNA demonstrated the presence of hydroxymethyluracil. Enzymatic degradation of pulse-labelled, nascent 0W-14 DNA followed by TLC suggested the presence of three or more novel nucleotides not found in uniformly labelled DNA samples. These observations were consistent with neutral CsCl analysis of pulse-labelled ØW-14 DNA. This DNA contained unusual heavy density components. ØW-14 ts and amber mutants were screened for defects in DNA replication or DNA modification by CsCl gradient and/or degradative analysis. Some DO mutants were identified. In addition, two DNA modification mutants were found. Am 42 made ØW-14 DNA containing lower-than-normal levels of a-putrescinylthymine and increased levels of thymine. Am 37 accumulated intermediates in a-putrescinylthymine biosynthesis. The conditionally lethal nature of the DNA modification lesion was demonstrated. DNA synthesis was adversely affected by this mutation but DNA precursor supplies were not impaired. Two atypical mononucleotides were purified from am 37 DNA. One was identified as hydroxymethyldeoxyuridylate. The second nucleotide was an acid-labile derivative of hydroxymethyldeoxyuridylate. Analysis of [6- ³H]-uracil and ³²PO₄ labelling ratios, chemical and enzymatic degradation and chromatographic analysis of this nucleotide demonstrated that it was the novel compound 5-(hydroxymethyl-0-pyro-phosphoryl)-deoxyuridylate (abbreviated to hmPPdUMP). 5-(hydroxymethyl-O-pyrophosphoryl)-uracil was shown to be a precursor of a-putrescinylthymine by in vitro modification of am 37 DNA with ØW-14 wild-type infected P. acidovorans cell-free extracts. In vitro modification confirmed that a-putrescinylthymine was formed at the polynucleotide level. ØW-14 DNA modification was not necessarily coupled to replication. The presence of hydroxymethyluracil in am 37 DNA agreed with the suggestion that hmPPura was formed by pyrophos-phorylation of hydroxymethyluracil in nascent DNA. HmPPdUMP had chromatographic properties similar to one of the compounds detected in pulse-labelled ØW-14 wild-type DNA. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

In eukaryotes, the genomic double-stranded DNA (dsDNA) coils around histones to form nucleosomes. Arrays of these nucleosomes bundle together to generate chromatin. Most DNA-related processes require interactions between chromatin-protected DNA and cellular machinery. Access of cell machinery to genomic DNA is partially regulated by the position and stability of nucleosomes, which may be influenced by changes in nucleosomal DNA. DNA is composed of adenine (A), guanine (G), cytosine (C), thymine (T) nucleotides and their derivatives. It has been shown that some C derivatives participate in directing multiple biological processes, and aberrant modification patterns are often linked to diseases. It has been proposed that T derivatives exhibit similar effects. This thesis focuses on elucidating the effect of naturally occurring DNA modifications on the properties of dsDNA and nucleosomes. dsDNA sequences systematically modified with various T derivatives were characterized using classical biophysical techniques to assess the effect of these DNA modifications. The results indicate that in the sequence context studied, 5-hydroxymethyluracil modifications destabilize dsDNA, while dense symmetrical 5-formyluracil (fU) modifications alter the dsDNA structure. These effects may provide clues to the differential protein recruitment observed in previous research. In vitro studies on nucleosome occupancy and stability revealed that 5-formylcytosine (fC) modifications have positive effects on nucleosome formation and stability compared to the unmodified counterpart by influencing the intrinsic biochemical and biophysical properties of the nucleosomes. These results provide casual links for the observation in vivo between fC and the increased nucleosome occupancy and positioning. In order to further understand the positional effect of fC on the nucleosomes, a method was developed for quick and reliable incorporation of C derivatives into dsDNA at desired positions. The positive effect of fC modifications on nucleosome occupancy and stability observed here has necessitated further studies to gain deeper insights into the biological functions of fC in the nucleosome context. Cryo-EM can be used to elucidate the structural foundation for the changes fC posts to nucleosome, and protein interacting assays will identify the cellular machineries specifically recruited/repulsed by fC-modified nucleosomes. The effect of DNA modifications elucidated by the above studies advances our understanding on the role that DNA modifications play in regulating cellular processes.

Graduation date: 1995

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 114-136). Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 170-179). Available also in a digital version from Dissertation Abstracts.

No description available.