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...

Expression of the red+ and gam+ genes of bacteriophage lambda in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these lambda functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2'-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red+ or gam+ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam+ or red+ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (sigma-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red+ products, beta protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD- ExoI- cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3' single-stranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.

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Alternative models for break-induced recombination predict different distributions of primary products. The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products. The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences. Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombination pathway of Escherichia coli. Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera phage by in vivo restriction, and the distribution of primary circular recombination products was determined. Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model. However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3 at the break. A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5 at the break (5 heteroduplexes) was observed at a later stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5 heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks.DNA double-stranded breaks (DSBs) initiate homologous recombination in bacteria, fungi, and higher eukaryotes (5-7, 16, 30, 40, 43, 51, 54, 55, 62). In Escherichia coli, DSBs play alternative roles in recombination, depending on the genotype of the strain and the structure of the recombination substrate. In recombination by the RecBCD pathway, ends serve as entry points for the RecBCD enzyme (28, 59, 60) that travels to the recombination site, where it generates a substrate for the RecA-mediated homologous pairing reaction (12,13,49). On the other hand, in recombination by the RecF pathway and the closely related RecE and Red pathway, ends may participate directly in homologous pairing (35,39,52,57,61,(63)(64)(65).Both ends flanking a DSB within a homology, or either one of them, may pair with the intact homolog. Concerted participation of both ends in homologous pairing is proposed by the DSB-repair (DSBR) recombination model (40). According to this model, exonuclease-mediated end processing yields...