Unbalanced deoxyribonucleic acid synthesis: Its role in X-ray-induced bacterial death

Abstract

A study has been made of the effects of unbalanced DNA synthesis on subsequent cell physiology and X-ray sensitivity of the polyauxotroph, Escherichia coli strain 15T-A-U- (thymine, arginine, and uracil dependent) with the following results: 1. 1. DNA replication, proceeding to completion in the absence of concomitant protein and RNA synthesis, resulted in a cell population with an enhanced DNA content and with increased resistance to the lethal effects of X-ray exposure. This resistance was expressed as an increase in the shoulder of the dose-survival curve and an increase in the mean lethal dose. 2. 2. Renewal of both protein and RNA synthesis after completion of unbalanced DNA synthesis reversed the resistant state. The presence of both arginine and uracil were required for maximum reversal. When only uracil was added, no change in sensitivity was seen. Addition of only arginine resulted in partial resensitization. 3. 3. Incubation of exponential cells in a medium lacking thymine as well as arginine and uracil led to thymine-less death as well as a sensitization to X-rays. This was characterized by a shift from sigmoidal to exponential killing. 4. 4. Substitution of 5-bromouracil for thymine during unbalanced DNA synthesis did not interfere with the completion of DNA synthesis. Buoyant density-gradient analysis in CsCl showed that only normal and hybrid DNA were present in such cells. Equilibrium centrifugation of DNA obtained from cells incubated with bromo[ 14 C]-uracil provided confirmation that only hybrid DNA was made during the unbalanced DNA synthesis period. Such cells did not exhibit enhanced resistance to X-rays, but to the contrary were more sensitive than the control population.