Abstract
Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.
Highlights
Thymine starvation causes irreversible toxicity and chromosomal damage, by unknown mechanisms
Significant New DNA Accumulation during T-starvation— it was always assumed that DNA replication is grossly inhibited in the absence of dTTP [2], the idea of ss-gap formation and repair behind T-starved replication forks (Fig. 1B) depends on the replication fork progress in T-starved cells
To understand the nature of chromosomal lesions leading to thymineless death” (TLD) in E. coli, we used physical techniques to characterize the chromosomal replication and degradation patterns during T-starvation in Recϩ cells, as well as in the recA and recBCD mutants
Summary
Thymine starvation causes irreversible toxicity and chromosomal damage, by unknown mechanisms. We described a novel method of DNA labeling during T-starvation using [32P]orthophosphoric acid [26] We employed this new DNA labeling technique, together with differential labeling for chromosomal fragmentation analysis with pulsed-field gels, as well as the marker frequency analysis with gene arrays, to show how the postulated single-strand gaps are converted into irreparable chromosomal lesions by toxic repair during T-starvation. We did it by examining the extent of initiation, replication, and chromosome damage in T-starved cells, in both recombinational repair proficient (Recϩ) and deficient (recA or recBCD mutants) cells
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