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

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Abstract
Near-ultraviolet photoproducts of l-tryptophan (TP) differentially inhibited deoxyribonucleic acid (DNA) replication in wild-type cells and uvrA, polA1, 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 × 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 high-molecular-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 polA1 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 polA1 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.