Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA, USA.
Molecular Microbiology (Impact Factor: 5.03). 05/2008; 68(1):66-74. DOI: 10.1111/j.1365-2958.2008.06133.x
Source: PubMed

ABSTRACT Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate--strand annealing or strand invasion--accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.

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    • "In Red-substituted E. coli lacking RecA, a non-replicating dsDNA phage chromosome introduced by infection recombines efficiently with an indigenous homology-bearing plasmid, if the plasmid is replicating and the non-replicating partner has a double strand break. The topology and kinetics of this recombination event suggest that the role of Red is to pair a ssDNA end with exposed ssDNA in a replication fork, and to induce a template switch, diverting the replisome onto the invading DNA [8]. "
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    ABSTRACT: The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability.
    PLoS ONE 06/2013; 8(6):e67440. DOI:10.1371/journal.pone.0067440 · 3.23 Impact Factor
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    • "This is surprising, given that according to the current mechanism, Lambda Exo must bind to a 5′ dsDNA end and degrade that strand in order for recombination to occur [21], [22]. Prior mechanisms for Red-mediated dsDNA recombination [24], [31] have similar implications, requiring Lambda Exo to degrade both 5′ ends of a dsDNA cassette. It is not immediately apparent how a cassette with both 5′ ends phosphorothioated could be processed by Lambda Exo and undergo recombination. "
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    ABSTRACT: Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3' ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.
    PLoS ONE 09/2012; 7(9):e44638. DOI:10.1371/journal.pone.0044638 · 3.23 Impact Factor
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    • "Recombination of the resulting single-strand products is carried out by b protein, either by annealing complementary single-stranded DNA (ssDNA) sequences or utilizing the exposed strand to invade a homologous duplex (Muniyappa and Radding, 1986; Rybalchenko et al., 2004). Genetic experiments suggest that b actually facilitates recombinant formation by a template switching mechanism during phage DNA replication (Poteete, 2008). An alternative exchange pathway is provided by the l Orf protein, which appears to supply a function equivalent to Escherichia coli RecFOR (Sawitzke and Stahl, 1992; Poteete, 2004; Cox, 2007). "
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    ABSTRACT: Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.
    Journal of Molecular Recognition 03/2011; 24(2):333-40. DOI:10.1002/jmr.1079 · 2.34 Impact Factor
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