Primary products of break-induced recombination by Escherichia coli RecE pathway.

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 lambda 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 larger 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.     

Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme.

The lambda Gam protein was isolated from cells containing a Gam-producing plasmid. The purified Gam protein was found to bind to RecBCD without displacing any of its subunits. Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single- and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase. When produced in vivo, Gam inhibited chi-activated recombination in lambda red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination. These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam. Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).     

Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway.

Interspecific recombination in conjugation between Escherichia coli and Salmonella typhimurium is several orders of magnitude lower than intraspecies recombination and is dependent on the RecA function. This low efficiency is due to a 20% divergence in the DNA sequence. The methyl-directed (mut H,L,S dependent) mismatch repair system appears to control the fidelity of homologous recombination; inactivating one of the Mut functions increases the interspecies recombination at least by 10(3)-fold. The interspecific recombination in mutS or mutL mutants is only approximately 10-fold lower than recombination in homospecific crosses as found after correction for the efficiency of mating and DNA transfer by zygotic induction experiments. The interspecific recombination is dependent on the RecABCD pathway: it was abolished in a recA mutant and decreased approximately 10(3)-fold in a recC mutant.     

Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.     

Effect of base pair mismatches on recombination via the RecBCD pathway

Summary The effect of base pair mismatches on recombination via the RecBCD pathway was studied in mutS and wild-type Escherichia coli, using substrates that contain single or multiple mismatches. Recombination between homologous DNA inserts in lambda phage and pBR322-derived plasmids forms phage-plasmid cointegrates that result from an odd number of crossovers. In the mutS host, when the sequence homology of a pair of 405 bp substrates decreased from 100% to 89%, the recombinant frequency decreased by about 9-fold, while in the wild-type host the decrease was about 240-fold. These results suggest that multiple mismatches can reduce recombinant frequencies by impeding the mechanism of recombination itself, and by provoking mismatch repair. Single mismatches in 31 bp substrates caused reductions in recombinant frequencies of 2-or 12-fold, depending on the location of the mismatch. However, unlike the reduction by multiple mismatches, the reduction of the recombinant frequencies by single mismatches was the same in both mutS and wild-type hosts. Thus a single match repair seems unable to act on single mismatches in very short homologies during recombination.     

Kinetics of ATP-stimulated nuclease activity of the Escherichia coli RecBCD enzyme

The RecBCD enzyme is an ATP-dependent nuclease on both single-stranded and double-stranded DNA substrates. We have investigated the kinetics of the RecBCD-catalyzed reaction with small, single-stranded oligodeoxyribonucleotide substrates under single-turnover conditions using rapid-quench flow techniques. RecBCD-DNA complexes were allowed to form in pre-incubation mixtures. The nuclease reactions were initiated by mixing with ATP. The reaction time-courses were fit to several possible reaction mechanisms and quantitative estimates were obtained for rate constants for individual reaction steps. The relative rates of forward reaction versus dissociation from the DNA, and the fact that inclusion of excess non-radiolabeled single-stranded DNA to trap free RecBCD has no effect on the nuclease reaction, indicates that the reaction is processive. The reaction products show that the reaction begins near the 3'-end of the [5'-32P]DNA substrates and the major cleavage sites are two to four phosphodiester bonds apart. The product distribution is unchanged as the ATP concentration varies from 10 microM to 100 microM ATP, while the overall reaction rate varies by about tenfold. These observations suggest that DNA cleavage is tightly coordinated with movement of the enzyme along the DNA. The reaction time-courses at low concentrations of ATP (10 microM and 25 microM) have a significant lag before cleavage products appear. We propose that the lag represents ATP-dependent movement of the DNA from an initial binding site in the helicase domain of the RecB subunit to the nuclease active site in a separate domain of RecB. The extent of reaction of the substrate is limited (approximately 50%) under all conditions. This may indicate the formation of a non-productive RecBCD-DNA complex that does not dissociate in the 1-2 s time-scale of our experiments.     

Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway

Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death. In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death.     

Chi-dependent intramolecular recombination in Escherichia coli.

Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5''-GCTGGTGG-3'') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.     

Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.

The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.     

Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system

Transformation of different Escherichia coli strains by linear dimers of pBR322 containing different tet alleles was investigated. Linear dimers transformed wild-type strains 0.1 to 1% as efficiently as circular dimers. In contrast, linear dimers transformed recBrecCsbcA strains, where the RecE recombination system is functional, as efficiently as circular dimers. The transformants contained plasmids that had a single recombinant monomer genotype, indicating that transformation was mediated by a recombination-dependent cyclization reaction. Altering the position of the double-strand break changed the frequency of recovering different recombination products, but had no effect on the frequency of transformation. Both the frequency of transformation and the production of Tcr recombinants were decreased by recE mutations, while recA and recF mutations were slightly stimulatory (twofold). Several recombination models consistent with these results are presented.     

Biochemistry of homologous recombination in Escherichia coli.

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.     

Plasmid effects on Escherichia coli metabolism

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.     

Plasmid Effects on Escherichia coli Metabolism

ABSTRACT:?

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.     

Role for the RecBCD recombination pathway for pilE gene variation in repair-proficient Neisseria gonorrhoeae

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec⁺ bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.     

DNA cloning by homologous recombination in Escherichia coli

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.     

Unusual stability of recombination intermediates made by Escherichia coli RecA protein.

The structure and stability of recombination intermediates made by RecA protein have been investigated following deproteinization. The intermediates consist of two duplex DNA molecules connected by a junction, as visualized by electron microscopy. Although we expected the structures to be highly unstable due to branch migration of the junction, this was not the case. Instead, we found that the intermediates were stable at 37 degrees C. At 56 degrees C, greater than 60% of the intermediates remained after 6 h of incubation. Only at higher temperatures was significant branch migration observed. This unexpected stability suggests that the formation of extensive lengths of heteroduplex DNA in Escherichia coli is likely to require the continued action of proteins, and does not occur via spontaneous branch migration. We show that heteroduplex DNA may be formed in vitro by ATP-dependent strand exchange catalysed by RecA protein or by the RuvA and RuvB proteins of E. coli.     

Plasmid stabilization of an Escherichia coli culture through cycling.

The problem of plasmid instability of fermentations that involve plasmid-bearing recombinant organisms is dealt with in this work. Previous theoretical work demonstrated that under certain conditions (where plasmid-bearing species are slower in responding to changes in the fermentation environment than the wild species) the washout of the plasmid-bearing species can be prevented. In the sequel, Weber and San showed that cycling the dilution rate can delay the washout of plasmid-bearing species for a plasmid-bearing Escherichia coli culture. This work shows that it is indeed possible to secure the presence of the plasmid-bearing species at all times through appropriate cycling.