Inhibition of Replication Gap Closure in Escherichia coli by Near-Ultraviolet Light Photoproducts of l -Tryptophan

Abstract

Near-ultraviolet photoproducts of L-tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polAl, and recA recB double mutants of Escherichia coli. Wild-type cells labeled in their DNA with [3H]thymidine in the presence of TP produced small pieces of DNA (7 x 106 daltons), which corresponded in size to late replicative intermediates of discontinuous DNA synthesis. Moreover, when TP was present, it took five times longer to chase the low-molecular-weight DNA pieces into highmolecular-weight DNA. The observation of replicative intermediates in the presence of TP, and their slow chase into high-molecular-weight DNA in the presence of TP, is strong evidence that TP stabilizes replication gaps in E. coli DNA. Although TP slowed DNA replication in wild-type cells, this effect was transient and DNA synthesis eventually resumed at a normal rate. However, in polAl and recA recB mutants, DNA synthesis was completely inhibited. Determinations of size and total counts of cells incubated in TP suggested that TP uncouples cell division from DNA replication in recA recB mutants, whereas these processes remain coupled in wild-type cells and in uvrA and polAl mutants. The slow chase of TP-stabilized pieces of DNA in the presence of TP suggested that the selective effect of TP on DNA synthesis and viability in repair-deficient mutants is a result of TP inhibition of replication gap closure.

Peroxide sensitivity of cold‐shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery

“…They proposed that in the absence of ligase activity cells were unable to rejoin DNA fragments produced during normal DNA replication which, in turn, allowed endonucleases to degrade DNA leading to cell death. Their hypothesis is not necessarily incompatible with ours because one reported effect of peroxide is to inhibit the repair of DNA strand breaks (Yoakum et al 1974).…”

Tryptophan photoproduct(s): sensitized induction of strand breaks (or alkali-labile bonds) in bacterial deoxyribonucleic acid during near-ultraviolet irradiation

“…To determine the reason for the genetic specificity of TP toxicity, we conducted experiments to test the effects of TP on deoxyribonucleic (DNA) metabolism. These studies demonstrated that TP blocks a step(s) required for the iapid closure of replication gaps (21) which occur during discontinuous semiconservate DNA replication (7). In addition, we found that TP inhibits one class of X-ray repair, without apparent blockage of other X-ray repair processes.…”

“…TP inhibits type III repair of X-ray-induced SS breaks without blocking type II repair of DNA SS breaks (20). Since TP blocks processes of DNA metabolism involved in the rejoining of DNA intermediates of repair and replication, we concluded that the dark toxicity of L-tryptophan photoproduct is probably a biological expression of the effect of TP on DNA metabolism (21).…”

“…The following strains were used in the experiments described below: W3110 wild type (thy) and P3478 (polAl, thy). These strains were originally derived from E. coli K-12, and their detailed genotypes and phenotypes have been described previously (21).…”

The recA+ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity

“…In Escherichia coli and Salmonella typhimurium, DNA is injured by hydrogen peroxide and some DNA repair-deficient strains are more sensitive to hydrogen peroxide than DNA repair-proficient strains (1,10,18,19,20). It has not been established whether these differences in sensitivity to hydrogen peroxide result from the various levels in the cell of the protecting enzymes catalase, peroxidase, or superoxide dismutase.…”