DNA synthesis and modification in ØW-14-infected Pseudomonas acidovorans
Creators: name: Maltman, Kirk Lee
Publisher: name: University of British Columbia
Year: 2010

description: 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.