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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

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