Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions

Abstract

Inactivation of RecBCD nuclease (exonuclease V) and SbcB nuclease (exonuclease I) in Escherichia coli K-12 diverts most of plasmid replication activity from circular monomer production to the synthesis of linear multimers. Linear multimer synthesis has been demonstrated in plasmids of diverse origins and copy numbers, including E. coli minichromosomes. The effect of dnaA, dnaB, recF, and recJ mutations on the rate of linear multimer synthesis in sbcB cells after gam inactivation of RecBCD nuclease was investigated. Results are consistent with the hypothesis that homologous recombination, but not activities at the plasmid origin of replication, is involved in initiation of linear multimer synthesis.Replication of ColEl-type plasmids is initiated at the 3' OH end of an RNA primer. After initiation, it proceeds unidirectionally by a theta-like mode of replication to yield circular plasmid monomers (26). Copy number is controlled at the primer formation stage in this system by a small RNA species, RNA I, which is transcribed from the DNA strand opposite to that encoding the primer (44).Recently, we (11) demonstrated another mode of replication for high-copy-number plasmids. This activity yields linear multimers rather than circular monomers. It is inhibited by RecBCD exonuclease and exonuclease I (sbcB enzyme) and depends on a functional RecA protein. Unlike normal replication, linear multimer synthesis is insensitive to plasmid-copy-number control mechanisms. Thus, inactivation of RecBCD nuclease in sbcB mutants leads to a rapid accumulation of plasmid linear multimers in the cell (11).Synthesis of linear plasmid multimers in recB recC sbcB mutants has been observed with derivatives of ColEl-type plasmids and with Xdv (11). It is of interest to determine whether it also affects other replicons such as low-copynumber plasmids and Escherichia troli minichromosomes. In this paper we address this question and determine the dependence of this mode of replication on functions which are involved in plasmid replication and recombination in wild-type cells. MATERIALS AND METHODSBacterial strains and growth conditions. Bacterial strains used are listed in Table 1. Plasmids carrying cultures were grown in L broth (32) supplemented with the appropriate antibiotics (100 ,ug of ampicillin per ml and 20 ,ug of kanamycin per ml). Cultures of temperature-sensitive strains and of mutants carrying pSF117 or pSF119 were grown at 28°C. c1857 from pSF117 or pSF119 was inactivated by a temperature shift to 37°C. DNA replication in dnaA46 or dnaB558 mutants was arrested by a temperature shift to 420C.Plasmids. Plasmids used in this study are listed in Southern hybridization analysis of plasmid molecular forms. Total DNA preparations (2 ,ug per lane) were subjected to electrophoresis for 16 h at 1.5 V/cm in TBE buffer on a 0.7% agarose gel (33). DNA was denatured, transferred to nitrocellulose filters, and hybridized to a 32P-labeled DNA probe by the Smith and Summers modification (41) of the Southern procedure (40). Labeled pSF117 was use...

DNA replication triggered by double-stranded breaks in E. coli: Dependence on homologous recombination functions

“…Thus, the rolling circle mode of DNA synthesis observed in this mutant is also likely to be RDR. It has been demonstrated that LPMs are efficiently generated in recBC sbcBC mutant cells in a manner dependent on the RecF pathway of homologous recombination (Silberstein and Cohen, 1987;Kusano et al, 1989). The multimer formation does not require the activity of plasmid replication origin (Silberstein and Cohen, 1987), suggesting that RDR is also induced in these mutant cells.…”

Completion of DNA replication in Escherichia coli

“…1D. plasmid instability similar to recD (25,35,36). Mutations in human homologs of these proteins are associated with genetic instabilities and impaired double-strand break repair (37).…”

Multicopy plasmid modification with phage λ Red recombineering

“…Since λ gam expression can cause plasmids to enter rolling circle replication by inhibiting the RecBCD exonuclease (Cohen and Clark, 1986;Silberstein and Cohen, 1987) we looked for Gam-dependent linear multimers of the plasmid using the method of Cohen and Clark (1986). With our standard 15-minute λ induction conditions for recombineering, we saw no evidence of Gam-induced high molecular weight linear multimers by Southern analysis (data not shown).…”