Thymine Starvation-induced Structural Changes in Escherichia Coli DNA. Detection by Pulsed Field Gel Electrophoresis and Evidence for Involvement of Homologous Recombination

Effect of thymine starvation on Escherichia coli DNA was investigated by using pulsed field gel electrophoresis combined with cell lysis in agarose gel. Post-lysis treatment with restriction enzymes generating relatively large fragments (NheI, SpeI or XbaI) revealed peculiar electrophoretic profiles specific for thymine-starved cells. Thus, a substantial portion of the DNA remained in the origin of electrophoresis (non-migrating DNA), and the amounts of the migrating fragments correspondingly decreased in an inverse relation to the map distance between the origin of replication (oriC) and each fragment. The formation of non-migrating DNA seems to depend upon the presence of replicated portions of the chromosome (sister duplexes), as judged by the effect of a preincubation at the non-permissive temperature in a dnaA(Ts) mutant. Electron microscopy showed that the non-migrating fraction of DNA was enriched with such structures as single-stranded tails or gaps and branchings with single-stranded arms. It was also found that the appearance of non-migrating DNA was highly dependent on the functional recA gene and moderately on certain RecF-family genes. These results strongly suggest that homologous recombination between sister duplexes is involved in the formation of the peculiar structures found in non-migrating DNA. A possible causal relationship between the formation of non-migrating DNA and viability loss (thymineless death) is also discussed.