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: 4.42). 05/2008; 68(1):66-74. DOI: 10.1111/j.1365-2958.2008.06133.x
Source: PubMed


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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    • "Stahl et al. [25] performed a detailed study to determine which mechanism was more likely, and recombination products from crosses of Lambda phage showed characteristics more consistent with annealing. Later studies demonstrating enhanced targeting of dsDNA recombineering to the lagging strand provided further support for the replication-fork annealing model [26] [27]. The most recent mechanism (Fig. 1), proposed by Mosberg et al. [28], hypothesized that replication-fork annealing occurred through a fully ss intermediate . "
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    ABSTRACT: Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, which can be divided into three main areas: (i) optimizing the oligo used for recombineering to enhance replication fork annealing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increased rate of annealing; and (iii) multiplexing recombineering targets and automation. These efforts have increased the efficiency of recombineering several hundred-fold. One area that has received far less attention is the problem of multiple chromosomes, which effectively decrease efficiency on a chromosomal basis, resulting in more sectored colonies, which require longer outgrowth to obtain clonal populations. Herein, we describe the problem of multiple chromosomes, discuss calculations predicting how many generations are needed to obtain a pure colony, and how changes in experimental procedure or genetic background can minimize the effect of multiple chromosomes.
    Biotechnology Journal 05/2013; 8(5). DOI:10.1002/biot.201200237 · 3.49 Impact Factor
    • "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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