Polymer Synthesis in Killed Bacteria: Lethality of 2′,3′-Dideoxyadenosine

Abstract

Philadelphia), MEEK1 JANSEN, AND SEYMOUR S. COHEN. Polymer synthesis in killed bacteria: lethality of 2',3'-dideoxyadenosine. J. Bacteriol. 92:565-574. 1966.-We studied the metabolic capabilities of cells that had lost the ability to multiply under a variety of lethal treatments. Cultures of a polyauxotrophic mutant of Escherichia coli strain 15 requiring thymine, arginine, uracil, and adenine for growth were killed to a few per cent survivors by several different methods. These treatments included streptomycin, thymineless death, D-arabinosyladenine (ara-A), and 2',3'-dideoxyadenosine (DDA). The killed cells were washed and supplied with complete media, and were compared with control cells with respect to the ability to incorporate thymine, arginine, and uracil. Cells killed with streptomycin in the absence of thymine were only partially inhibited in deoxyribonucleic acid (DNA) synthesis; they were markedly inhibited in synthesis of ribonucleic acid (RNA) and protein. Cells that had suffered thymineless death were essentially uninhibited in DNA synthesis, partially so in RNA synthesis, but extensively inhibited in protein synthesis. Killing by ara-A did not prevent DNA synthesis, but markedly inhibited RNA and protein synthesis. The lethality of DDA was studied in the presence of exogenous adenosine; lethality was partially prevented by deoxyadenosine. Dideoxyadenosine was similar to ara-A and thymineless death in killing in a pattern in which RNA and protein synthesis continued while DNA synthesis was inhibited. Cells killed by DDA, however, were markedly inhibited in subsequent thymine incorporation, unlike cells killed by the other methods. In addition, at this time, the DDA-killed cells were more inhibited in incorporation of arginine than of uracil. DDA also potentiated thymineless death; when cells were killed rapidly by the combined treatment, only the ability to synthesize DNA was lost irreversibly. This agent (DDA) may permit the detailed study in E. coli of the relation of DNA synthesis to numerous phenomena, such as genetic recombination, sequential transcription, and the number and distribution of chromosome breaks.

Effects of 2′,3′‐dideoxynucleosides on mammalian cells and viruses

“…The conversion of ddThd to ddTTP is also negligibly slow in E. coli (Atkinson et al, 1969), but d a d o is converted to ddATP in E. coli at a sufficiently rapid rate to allow irreversible inhibition of cellular DNA synthesis (Doering et al, 1966;Toji and Cohen, 1969). This observation raises the possibility that one of the other three ddNs might show more pronounced effects on cells or viruses than those demonstrated by ddThd.…”

DNA chain termination by 2',3'-dideoxythymidine in replicating mammalian cells

“…The thymidine triphosphate analog 2/,3'-deoxythymidine triphosphate can be used as a substrate by DNA polymerase I of Escherichia coli and incorporated into growing DNA chains in vitro, whereupon the chains terminate because of the lack of the 3'-hydroxyl group necessary for chain continuation (Atkinson et al, 1969). The nucleoside analog 2',3'-dideoxyadenosine irreversibly inhibits DNA synthesis in E. coli (Doering et al, 1966). In the present study an attempt has been made to utilize the chain-terminating function of 2/,3/-dideoxythymidine (ddT)* 1 *in mammalian cells to ascertain its possible value for analysis of replication in complex systems.…”

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

“…The compound had been tested because the absence of a 3'-hydroxyl group indicated that the derived nucleotide might terminate deoxyribonucleic acid (DNA) chains. Indeed, the metabolic lesion observed in E. coli was a relatively specific irreversible inhibition of DNA synthesis (3), a result suggesting the validity of the proposed mechanism of toxicity. More recently, we showed that the triphosphate of this dideoxynucleoside does in fact terminate polydeoxynucleotide chains in vitro when a purified DNA polymerase derived from E. coli is used (16).…”

“…The lethality of 2',3'-dideoxyadenosine to several strains of Escherichia coli was demonstrated in 1966 (3). The compound had been tested because the absence of a 3'-hydroxyl group indicated that the derived nucleotide might terminate deoxyribonucleic acid (DNA) chains.…”