Role of DNA Replication and Repair in Thymineless Death in Escherichia coli

Abstract

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.Thymineless death (TLD), the phenomenon in which exponentially growing cells starved for thymine lose viability, has been researched for over five decades, but the molecular mechanism remains an enigma. Thymine starvation halts DNA synthesis; Maaløe and Hanawalt (9) proposed a connection between stalled replication forks and TLD in Escherichia coli after showing that only those cells actively replicating DNA when thymine was removed underwent TLD (8). To test the role of replication in TLD, we examined the impact of various concentrations of hydroxyurea (HU) on the viability of E. coli cells starved for thymine. HU specifically inhibits replication by preventing the function of ribonucleoside diphosphate reductase, thus starving the cell for all four deoxynucleotides. At high concentrations, HU also significantly inhibits transcription by an unexplained mechanism, but at low concentrations, the effects of HU appear to be largely limited to inhibition of DNA replication (14).HU exposure affected TLD in a concentration-dependent manner. The protection against TLD afforded by HU increased with increasing concentrations of HU. The thyminerequiring E. coli strain HL813 (all strains used in this study are listed in Table 1) was grown overnight at 37°C in Difco Davis minimal medium containing 0.4% glucose and 0.1% Casamino Acids (DM medium) supplemented with 10 g/ml thymine. The overnight culture was diluted in the same medium and grown at the same temperature with shaking. During the exponential phase (10 6 to 10 8 cells/ml), cells were collected on a Millipore filter (pore size, 0.45 m) and washed and resuspended in DM medium without thymine. HU was added to the specified bacterial cultures at the designated final concentration, and at the indicated time intervals 0.01-ml aliquots were removed, appropriately diluted, and plated on DM agar plates supplemented with 10 g/ml thymine for the determination of viability. When 200 mM HU was added to thymine-starved cells, viable cell counts remained constant over a 4-h time period (Fig. 1a). However, TLD was observed at lower concentrations of HU, albeit at a slightly lower rate than in the control without HU (Fig. 1b and c). At higher HU concentrations, such as 200 mM, RNA synthesis was greatly reduced (Fig. 2a). Since inhibition of RNA synthesis protects against TLD (4, 6, 10), it is likely that the lack of TLD at 200 mM HU is mostly due to inhibition of RNA synthesis. However, inhibition of DNA synthesis could also be involved in the TLD protection, because DNA synthesis was completely inhibited in the presence of 200 mM HU (Fig. 2b). On the other hand, although DNA synthesis was inhibited more than 90% at the lowest concen...

Double-Strand Break Generation under Deoxyribonucleotide Starvation in Escherichia coli

“…4). Recently, Morganroth and Hanawalt have observed that TLD occurs after Hu addition (21). According to our proposal, a recovery of TLD could be expected after Hu addition, but it has to be pointed out that our prediction would be only in the case that the stalled fork underwent RFR under these conditions.…”

“…That these DSBs occurred at or near the replication forks would explain the connection between replication forks stalled by thymine starvation and TLD proposed by Maaloe and Hanawalt in the 1960s after showing that only those cells actively replicating DNA when thymine was removed underwent TLD (13,17). Our proposal is that this connection is not by the replication process itself, as it has been shown not to be required (21), but through the fate of the stalled replication forks when thymine is removed.…”

“…According to our proposal, a recovery of TLD could be expected after Hu addition, but it has to be pointed out that our prediction would be only in the case that the stalled fork underwent RFR under these conditions. In the abovementioned work, Hu addition was performed at the same time as the culture was thymine starved (21), and under these conditions, the fate of the stalled forks is, at this moment, unknown. Nevertheless, our results agree with the more general observation of the Morganroth and Hanawalt work that active DNA synthesis is not required for TLD, as we suggest that TLD would be related to the fate of the stalled replication under thymine starvation rather than to the occurrence of the replication process itself.…”

Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

“…mazEF has been shown to be responsible for programmed cell death under a wide variety of seemingly unrelated stressful conditions (e.g., short-term antibiotic treatments, high temperature, and oxidative shock) by the group of Engelberg-Kulka (14,18,31). However, chromosomally encoded TA system-dependent programmed cell death was not observed in work by other groups (5,7,22,25,26,33,34) and was recently ruled out by our group (38). The other mainstream hypothesis is that amino acid starvation induces chromosomally encoded TA system-dependent bacteriostasis.…”

Analysis of Chromosomal Replication Progression by Gel Electrophoresis

“…The nmDNA was characterized as having single-stranded tails or gaps and branching with single-stranded arms. TLD has been related to DNA replication ; nevertheless, ongoing replication does not appear to be required for TLD as same lethality is observed under thymine starvation in the presence of hidroxyurea (Morganroth & Hanawalt, 2006), an inhibitor of the DNA synthesis. By contrast, TLD is suppressed by the addition of rifampicin or cloramphenicol, both inhibitors of the new initiation events of the E. coli chromosomal DNA (Hanawalt, 1963).…”