THE ABORTIVE REPLICATION OF ΦX174 DNA IN A RECOMBINATION-DEFICIENT MUTANT OF Escherichia coli

Abstract

There is reason to believe' that host-cell enzymes catalyze several of the steps in the replication of the single-stranded DNA of the virus 4bX174, particularly the conversion of the single-stranded DNA to the double-stranded replicative form (RF)l and, perhaps, the subsequent replication of the RF. Hence one could expect to find bacterial mutants in which the 4X DNA cannot be replicated. Such mutants would more clearly define host-controlled steps in the viral life cycle. In addition, one might be able to use the viral DNA as a probe of the fate of nucleic acids in the mutant cell; that is, it should be possible to understand any concomitantly occurring deficiency in cellular nucleic acid metabolism in terms of the experimentally analyzable defect in 4X DNA replication.Because of the potentially informative nature of mutants with altered patterns of DNA metabolism, and because of our interest in 4X replication, we sought mutants of a 4IX-sensitive strain that would adsorb but not replicate the virus. We report here the initial characterization of a bacterial mutant (called REP-) that does not allow the replication of 4X RF and additionally has a reduced capacity for bacterial recombination. It is representative of a heretofore undescribed class of recombination-deficient mutants.Materials and Methods.-Solutions and media: TKCaB contains 10 gm Bacto-Tryptone (Difco), 5 gm KCl, and 0.015 M CaC12 in 1 liter of distilled H20. When growing thyminerequiring cells, 10 Ag/ml thymidine is added. Tryptone broth contains, per liter: 10 gm tryptone, 5 gm NaCl, 0.01 M MgSO4, and 20 mg thymidine. m3XD contains 3 gm NH4Cl, 0.3 gm MgS04 7H20, 15 gm Difco Casamino acids, 24 ml glycerol, 1 mg gelatin, 0.9 gm KH2P04, 2.1 gm

All motors have to decide is what to do with the DNA that is given them

“…The Rep helicase from Escherichia coli is a 3′-5′ SF1 DNA helicase that is involved in replication restart (55) and is required for replication of some bacteriophages (56). Monomers of Rep are capable of translocating from 3′ to 5′ along ssDNA with a macroscopic rate that varies with solution conditions (51).…”

Role of the Rep Helicase Gene in Homologous Recombination in Neisseria gonorrhoeae

“…It has been demonstrated in E. coli that rep mutants display decreased conjugation frequency when rep is absent in the recipient strain (7,8). We tested whether rep might also be involved in the homologous-recombination-mediated process of DNA transformation in N. gonorrhoeae.…”

“…Escherichia coli rep mutants were originally identified by their inability to support phage replication (4, 7). Rep has also been implicated in chromosomal replication (6,24,25), DNA repair, and homologous recombination (4,7,8) A direct link between replication and recombination has been demonstrated for E. coli (21, 36) in a process called replication restart. During this process, replication can be resumed from recombination intermediates formed at stalled replication forks.…”

“…Escherichia coli rep mutants were originally identified by their inability to support phage replication (4, 7). Rep has also been implicated in chromosomal replication (6,24,25), DNA repair, and homologous recombination (4,7,8) …”

“…It has been proposed that the two DNA molecules that recombine during antigenic variation may arise just after the genome is replicated (16,17), suggesting a possible link between antigenic variation and DNA replication. Since the Rep helicase plays a role in replication and recombination in E. coli (7,8), we tested whether the gonococcal rep gene is involved in pilin antigenic variation.…”