No abstract

Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA-, dnaBts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA+ strains. Repair synthesis does not require the uvrA gene product.

The antibiotic bleomycin stimulates deoxyribonucleic acid (DNA) synthesis in toluene-treated Escherichia coli cells. The increase in synthesis is linear with bleomycin concentration. Bleomycin-stimulated DNA synthesis is independent of replication and dependent on DNA polymerase I. Replication is spared as the DNA polymerase I-dependent DNA synthesis increases. Bleomycin does not appear to have any effect on purified E. coli DNA polymerases I or II. Our results suggest that bleomycin causes nicking of the bacterial chromosome with subsequent DNA synthesis catalyzed by DNA polymerase I.Bleomycin was first isolated and purified by Umezawa and co-workers in 1966 (30, 31, 33) and identified as a glycopeptide with a molecular weight of -1,600. Initially, it was investigated as both an antimicrobial and antitumor agent (22,25), and it has proved to be effective in the control of Hodgkin disease (34). Furthermore, the drug inhibits the growth and reduces the size of rat mammary carcinomas induced by 7,12-dimethylbenz-(a)-anthracene (29). Bleomycin inhibits deoxyribonucleic acid (DNA) synthesis in Escherichia coli, Ehrlich ascites cells, and HeLa cells (25). Fujimura et al. (6) and Haidle et al. (10) have shown that bleomycin can induce a prophage from a stable lysogen growing in liquid culture. Subsequent work by others indicates that bleomycin apparently causes single-stranded breaks in DNA, as determined by the sedimentation of bleomycintreated DNA in neutral or alkaline sucrose density gradients (7,9,21,26). Muller et al. (22) showed that bleomycin inhibits DNA-dependent DNA polymerase from Rauscher murine leukemia virus. Two different laboratories (11, 21) have reported that bleomycin is able to cause the loss of a base, leaving an aldehyde. Thus it is possible that bleomycin exerts its antibiotic effect by alterations in DNA metabolism.Since the evidence indicated that bleomycin causes DNA strand scissions, we decided to test whether the drug would have any direct effect on DNA synthesis. For these experiments we chose the toluene-treated cell system. This in vitro DNA-synthesizing system exhibits replicative and enzymatic repair synthesis and allows use of cells that are defective in one or more of the enzymes of DNA synthesis (18).We have examined the effect of bleomycin on DNA synthesis in toluene-treated E. coli and find that it stimulates DNA synthesis of a nonreplicative type. Our results indicate that an active DNA polymerase I must be present for this stimulation of synthesis to occur in the toluene-treated cell. In the presence of adenosine 5'-triphosphate (ATP) replication persists. Since bleomycin induces a polymerase I-mediated, nonreplicative type of synthesis, the drug should be a useful probe of DNA repair mechanisms in toluene-treated cells.MATERIALS AND METHODS Materials. Bleomycin (lot no. 72L570) was kindly provided by Alexander Lane of Bristol Laboratories. 3H-labeled deoxythymidine 5'-triphosphate (dTTP) was obtained from New England Nuclear Corp. and a-32P-labeled deoxycytidine 5'-tripho...