DNA replication is regulated in response to environmental constraints such as nutrient availability. While much is known about regulation of replication during initiation, little is known about regulation of replication during elongation. In the bacterium Bacillus subtilis, replication elongation is paused upon sudden amino acid starvation by the starvation-inducible nucleotide (p)ppGpp. However, in many bacteria including Escherichia coli, replication elongation is thought to be unregulated by nutritional availability. Here we reveal that the replication elongation rate in E. coli is modestly but significantly reduced upon strong amino acid starvation. This reduction requires (p)ppGpp and is exacerbated in a gppA mutant with increased pppGpp levels. Importantly, high levels of (p)ppGpp, independent of amino acid starvation, are sufficient to inhibit replication elongation even in the absence of transcription. Finally, in both E. coli and B. subtilis, (p)ppGpp inhibits replication elongation in a dose-dependent manner rather than via a switch-like mechanism, although this inhibition is much stronger in B. subtilis. This supports a model where replication elongation rates are regulated by (p)ppGpp to allow rapid and tunable response to multiple abrupt stresses in evolutionarily diverse bacteria.
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.
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