Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli.

Abstract

Recombinant DNA molecules of phage X formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity.recA -cells were found to be capable of forming recombinant A phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the addition of rifampin. The introduction of the rifr mutation into host bacteria made their recombination activity rifaimpin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recAindependent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-0 regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination. In order to understand the roles of Escherichia coli functions in genetic recombination, we have developed a system that has enabled us to study recombination separated from DNA replication, transcription, translation, and progeny formation (1). E. coli was mixedly infected with two mutants of A phage in the presence of thloramphenicol and rifampin, and the recombinant DNA formed within the cell was detected by in vitro packaging. In this. system, the recA gene product was required for the formation of recombinant DNA molecules, in agreement with studies on recA mutations by other investigators (2). During the course of this study, we encountered an unexpected observation that recA mutations did not block completely the formation of recombinant X DNA when mixed infection was carried out in the presence of chloramphenicol without addition of rifampin. This fact suggested that the recombinant DNA was formed in recA -cells by a rifampin-sensitive mechanism. In the present study, we show that RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) is involved in this recA-independent pathway of recombination and that the transcription process per se plays an essential role in this recombination. MATERIALS AND METHODSBacterial and Phage Strains. The Escherichia coli K-12 and bacteriophage A strains used are listed in Tables 1 and 2, respectively.Media and Chemicals. Media, buffer, and chemicals used are generally those described previously (1). The final concentrations of chloramphenicol (Sankyo) and rifampin (B grade, Calbiochem) are 150 and 200 ,ug/ml, respectively, unless otherwise stated.Mixed Infection. The procedure was the same as that described previously (1). In brief, bacteria were incubated with inhibitor(s) (chloramphenicol only or chloramphenicol and rifampin) in A dilution buffer [10 mM MgSO4/10 mM Tris-HCl (pH 7.4...

Transcription-Associated Genome Instability

“…The first evidence that transcription influences recombination comes from a study in which recombination between two λphages was shown to occur at transcribed sites independently of the HR protein RecA. 105 Importantly, these events required the activity of E. coli RNAP, as shown using the RNAP inhibitor rifampicin. Furthermore, the recombination sites were shown to be extended to new regions in rho termination mutants.…”

Transcription and Recombination: When RNA Meets DNA

“…The first evidence of TAR comes from l-phage studies showing that recombination occurs in transcribed DNA regions and depends on Escherichia coli RNA polymerase (RNAP) activity but not on RecA (Ikeda and Kobayashi 1977;Ikeda and Matsumoto 1979). Probably, the most influential reports on TAR were those showing the identification of the recombinant DNA (rDNA) sequence HOT1 as a hot spot of recombination in Saccharomyces cerevisiae (Keil and Roeder 1984), and the following demonstration that the ability of HOT1 to stimulate Rad52-dependent ectopic recombination of non-rDNA sequences depends on the RNAPI activity (Voelkel-Meiman et al 1987;Stewart and Roeder 1989).…”

Role of R loops in recA-independent homologous recombination of bacteriophage lambda

“…An amber phage, K imm434 cI Daml5 FIam96B b538 red3, was added to one of the tubes in the presence of chloramphenicol, and another amber phage, K imm434 cI Sam7 RamS int6 red3, was added to the other tube under the same conditions (separate infection). After incubation with the drug, the cultures were mixed, and their DNAs were extracted as described previously by Ikeda and Kobayashi (9). Samples of the DNA preparation were packaged in vitro in a lysate of E. coli recA, NS428, and NS433.…”

“…Recombination of bacteriophage X is known to be mediated by functions of Escherichia coli genes, called rec, as well as viral genes, called red and int (6,7,18,20). We have previously shown that recA-independent homologous recombination of phage X takes place in the E. coli system, in which cells were jointly infected with phage X in the presence of chloramphenicol, a protein synthesis inhibitor, and intracellular X DNA was extracted and packaged in vitro to detect recombinant phage particles (9). The recombination takes place independently of Int and Red functions and is promoted by DNA-dependent RNA polymerase.…”