Sources of thymidine and analogs fueling futile damage-repair cycles and ss-gap accumulation during thymine starvation in Escherichia coli

Abstract

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of action of popular antibacterial and anticancer drugs, yet the mechanisms of TLD are still unclear. TLD comprises three defined phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding key to TLD pathology. We propose that a limited source of endogenous thymine maintains replication forks through the resistance phase. When this source ends, forks undergo futile break-repair cycle during the RED phase, eventually rendering the chromosome non-functional. Two obvious sources of the endogenous thymine are degradation of broken chromosomal DNA and recruitment of thymine from stable RNA. However, mutants that cannot degrade broken chromosomal DNA or lack ribothymine, instead of shortening the resistance phase, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal DNA accumulation during the resistance phase is negated during the RED phase, suggesting futile cycle of incorporation and excision of wrong nucleotides. We tested incorporation of dU or rU, finding some evidence for both, but DNA-dU incorporation accelerates TLD only when intracellular [dUTP] is increased by the dut mutation. In the dut ung mutant, with increased DNA-dU incorporation and no DNA-dU excision, replication is in fact rescued even without dT, but TLD still occurs, suggesting different mechanisms. Finally, we found that continuous DNA synthesis during thymine starvation makes chromosomal DNA increasingly single-stranded, and even the dut ung defect does not completely block this ss-gap accumulation. We propose that instability of single-strand gaps underlies the pathology of thymine starvation.

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity

“…Our unsuccessful attempts to aggravate TLD by artificially increasing the number of replication forks in the chromosomes of prestarved cells suggested either general TLD independence of the number of replication forks or at least TLD independence of the number of preexisting forks. According to the latter idea, only the forks that are induced during T starvation make the chromosome vulnerable to TLD, the idea being supported by the initiation spike in response to T starvation, followed by the disappearance of overinitiated DNA during TLD (9,16). The initiation spike in response to T starvation is a natural reaction of cells to inhibition of DNA synthesis during normal cell mass increase (27,36).…”

“…While we are in the process of evaluating these observations and ideas, a somewhat less dramatic loss of the chromosomal DNA during TLD at 37°C (Fig. 7F) turned out to be independent of the RecBCD enzyme, the main linear DNA degradation activity in E. coli (9), making the overall scenario unlikely. Also, it remains to be tested whether this dramatic chromosomal DNA loss is the cause or the consequence of cell death during T starvation.…”

“…When just discovered, the phenomenon of thymineless death was thought to be due to an unbalanced growth, an inability to replicate DNA in otherwise growing cells (2,6). However, a recent experimental testing of two specific rebalancing strategies as explanations for the resistance phase turned up negative (9). Interestingly, populational alignment by chloramphenicol treatment or amino acid starvation of auxotrophs promoted the conclusion that cells with no replication forks in their chromosomes develop immunity against thymineless death (11,12).…”

“…Since the evolution of the absolute origin amount did not reveal the reason for the increasing ori/ter ratio in the thyA dnaA mutant, we looked at the evolution of the absolute terminus amount, expecting more modest changes, observed previously both at 28°C (16) and at 37°C (9). In the presence of dT, the absolute terminus amounts in the thyA dnaA(Ts) mutant at 42°C behaved as expected, climbing steadily and then plateauing at 2-fold the original amount ( Fig.…”

Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis

“…Since actively growing cells continuously require a lot of dT to replicate chromosomal DNA, existing replication forks were inferred to be the points of TLD pathology (7,8,(13)(14)(15). Indeed, T-starvation severely inhibits chromosomal DNA replication (15) and is associated with accumulation of single-stranded DNA, suggesting generation of single-strand (ss) gaps by attempted replication in the absence of dT (7,16). These ss-gaps induce the SOS response (7,8,17), which contributes to the pathology of TLD by induction of the SulA cell division inhibitor (8).…”