DNA replication initiation as a key element in thymineless death

Abstract

Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles (n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase.