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.
A long-standing enigma in the fields of DNA repair and cancer chemotherapy is why it is that cells starved of the base thymine die rapidly. This process, called thymineless death (TLD), is conserved in bacterial, yeast, and human cells and is the mode of action of important cancer chemotherapeutic drugs. Tumors that become resistant to those drugs have ceased to die from TLD. Despite its ubiquity, importance, and having been studied for more than 50 years, the mechanism(s) of TLD remained elusive. Here 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 (or SOS) 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. In cells undergoing TLD, we observe blocked replication of the E. coli chromosome followed by loss of DNA near the replication origin then terminus. This implies that much of TLD results from an irreversible cell-cycle checkpoint that blocks cell division when single-stranded DNA (the SOS–inducing signal) accumulates and that the rest results from DNA destruction, models for which are presented.