doi:10.1016/S0378-1097(01)00242-7 
Copyright © 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
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What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function
Anthony R. Poteete
, 
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
Received 20 March 2001;
revised 14 May 2001;
accepted 15 May 2001
Available online 2 July 2001.
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Abstract
Recent studies have generated interest in the use of the homologous recombination system of bacteriophage λ for genetic engineering. The system, called Red, consists primarily of three proteins: λ exonuclease, which processively digests the 5′-ended strand of a dsDNA end; β protein, which binds to ssDNA and promotes strand annealing; and γ protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a ‘hyper-rec’ state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.
Author Keywords: Bacteriophage λ; Homologous recombination; Genetic engineering; λ Red system
Article Outline
- 1. Introduction
- 2. Components of the Red recombination system
- 3. Mechanisms of Red-mediated λ recombination
- 4. Red-mediated recombination out of the phage context
- 5. Use of Red in genetic engineering
- 6. Modulation of Red-mediated recombination by other λ proteins
- 7. Biological function of Red
- Acknowledgements
- References
Fig. 1. Genomic organization of the lambdoid bacteriophages. The order of genes shown is that of the integrated prophage, with ‘up’ corresponding to ‘left’ in the usual map representation. The major promoters of the phage lytic cycle are indicated by arrows. Two types of homologous recombination systems, consisting of orthologs of the λ Red genes, or of the P22 erf-abc genes, have been identified [30 and 32].
Fig. 2. Pathways of Red-mediated double-strand break repair. Invasion (left) is efficient only in the presence of RecA, whereas annealing is efficient in the absence of RecA [12].
Fig. 3. Chromosomal gene replacement by a linear dsDNA fragment, either released from an infecting phage by the action of an endogenous restriction endonuclease, or introduced directly into the cell by electroporation.
Fig. 4. Red-mediated replacement of lac with cat in the chromosome of E. coli recGΔ. The depicted molecular events would have to take place on both sides of the cat gene to generate a recombinant; for clarity, only one side is shown. The double-stranded end initiates recombination. λ exonuclease processively digests the 5′-ended strand, leaving a 3′-ended single-stranded tail. The combined action of RecA and the λ β protein mediates invasion of the 3′-ended strand into an unbroken homologous duplex. RecFOR is a key participant in the overall reaction pathway; its properties suggest that it is involved either in loading or unloading RecA (and β?) from the joint molecule. Once a three-stranded junction is formed, the crossed strands are subject to RuvAB and/or RecQ helicase-driven branch migration, resulting in a Holliday junction, which can be resolved by RuvC into a recombinant molecule.
Abstract
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