Abstract
Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.
Highlights
Thymineless death (TLD), the rapid loss of viability in cultures deprived of thymine, occurs in E. coli, yeast and human cells
We show that a large fraction of TLD requires RecA, the central protein in homologous recombinational (HR) DNA repair, and activation of the bacterial DNA–damage response, which RecA controls
We find that of the 40 or so proteins upregulated during an SOS response, SulA, an inhibitor of cell division, accounts for most of how SOS–activation causes TLD
Summary
Thymineless death (TLD), the rapid loss of viability in cultures deprived of thymine, occurs in E. coli, yeast and human cells (reviewed [1]). Whereas 5-FU kills cells both TLD-dependently and TLD-independently (reviewed [2]), newer drugs are being developed that target thymidylate synthase [3]. Work by Sat et al suggested that TLD in E. coli was a form of cell suicide induced by the MazF toxin gene, an RNase that can induce cell death under various stresses coincident with destruction of mRNAs [4,5] by a mechanism not fully understood. Though intriguing, this is probably not the full story of TLD. Whereas inhibition of transcription by various drugs relieved TLD [6,7,8], MazF is repressed under active transcription by the presence MazE anti-toxin, and becomes available when transcription is inhibited and MazE is degraded [9], such that inhibiting transcription would have been expected to exacerbate TLD (discussed [8])
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