Phases of thymineless death inEscherichia coli 15 TAU

On the basis of the course of loss of colony-forming ability it was possible to disting guish at least three phases of thymineless death in a culture ofEscherichia coli 15 TAU starved in a thymine-free medium enriched with arginine and uracil. The main differences between the individual phases were: (a) half-time of the loss of colony-forming ability; (b) only in the first phase was thymineless death reversible; (c) beginning in the second phase, part of the cells lysed after thymine was added to the liquid medium or when cells were plated on agar with complete medium; (d) lysis was much slower in the third phase than in the second; (e) in the presence of caffeine the course of the first and second phase was not affected but cells did not die in the third phase. Cells surviving in the third phase in the presence of caffeine are probably those which did not die even in medium T^?A^?U^? (Maaløe & Hanawalt, 1961). Transition from one phase to another was caused neither by change in the composition of medium during thymine starvation nor by heterogeneity of this culture from a genetical point of view.     

Kinetics of thymineless death in Escherichia coli 15 as measured by single-cell observations.

Kinetics of thymineless death for Escherichia coli 15 TAU-bar from plating on solid medium were compared with those from direct observations of single cells under a microscope. The latter method did not involve any physical change of the medium. The kinetics obtained for the two methods were identical. This rules out the assumption that in E. coli 15 TAU-bar death from the thymine deprivation is directly associated with the plating procedure.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

Role of DNA replication and repair in thymineless death in Escherichia coli

Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.     

Escherichia coli RecQ helicase: a player in thymineless death

DNA helicases of the RecQ family are distributed among most organisms and are thought to play important roles in various aspects of DNA metabolism. The founding member of the family, RecQ of Escherichia coli, was identified in a study aimed at clarifying the mechanism of thymineless death, a phenomenon underlying the mechanism for the cytotoxicity of the anticancer drug 5-fluorouracil. The present article is concerned solely with E. coli RecQ and tries to offer an integrated picture of the past and present of its study. Finally a brief discussion is given on how RecQ is involved in thymineless death.     

Pathways of resistance to thymineless death in Escherichia coli and the function of UvrD

Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies.