Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Thomason, Lynn C.; Court, Donald L.; Bubunenko, Mikail; Costantino, Nina; Wilson, Helen Rose; Oppenheim, Amos B.

doi:10.1002/0471142727.mb0116s62

Cited by 61 publications

(86 citation statements)

References 12 publications

(11 reference statements)

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“…Strains were induced at 42°for 15 min to express Red functions and immediately made electrocompetent as described (27,28). A saturating level of each ssDNA oligo (5 pmol) was used per electroporation.…”

Section: Methodsmentioning

confidence: 99%

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Costantino

Court

2003

Proc. Natl. Acad. Sci. U.S.A.

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263

359

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Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage -encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20 -70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence. H omologous recombination mediated by Red has been used as a genetic tool to modify the bacterial chromosome with linear double-stranded DNA (dsDNA) (1-6). In addition to linear dsDNA, single-stranded DNA (ssDNA) oligonucleotides (oligos) have been used to modify the chromosomes of both yeast and Escherichia coli (7-9). In yeast, the functions are not yet defined that allow oligo recombination; however, in E. coli, the bacteriophage Red recombination system is involved (9).The Red system includes the Gam, Exo, and Beta proteins. Whereas Red-mediated recombination between linear duplex DNA and the bacterial chromosome requires all three functions (1, 3, 10), recombination with ssDNA oligos requires only the Beta protein (9). Beta protein binds stably to ssDNA (11) Ͼ35 nt in length (12), protects it from single-strand nuclease attack (13,14), and promotes annealing to complementary ssDNA (13-15).Beta binds ssDNA from 3Ј to 5Ј (13, 16) but does not bind directly to dsDNA (13,14). However, after Beta generates dsDNA by annealing two complementary ssDNAs, it remains tightly bound to the annealed dsDNA (13,14,16). As it anneals strands to form the dsDNA, it generates DNA filaments similar to those formed by . This annealed dsDNA-Beta complex is resistant to DNase I and is much more stable than the ssDNA-Beta complex (16).Although Beta can catalyze single-strand annealing, it cannot promote strand invasion of a duplex DNA with a homologous ssDNA during recombination (16,18). Therefore, Betamediated recombination with ssDNA is likely to occur by annealing with transiently single-stranded regions of the chromosome. Initial results suggest that Beta-dependent ssDNA recombination may occur at the DNA replication fork. When either of the two complementary ssDNA oligos...

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Section: Methodsmentioning

confidence: 99%

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Costantino

Court

2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

263

359

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“…Constructions were based on a previously described method (Nielsen et al 2006) in E. coli VH1000, a ΔlacZ MG1655 derivative described previously (RLG3499) (Gaal et al 1997). Briefly, parS sites (binding sites for ParB-GFP derivatives) were inserted at the positions shown in Supplemental Figure S1 by double-stranded recombineering (Thomason et al 2007) using the primers listed in Supplemental Table S1. DNA fragments were amplified from genomic DNA from strain RLG7419 (FHC2973) (Nielsen et al 2006) with primer pairs containing 40-45 nt of homology with the intended chromosomal site of insertion followed by 20-22 nt of homology with the parS-kanamycin (kan) or parS-chloramphenicol (cam) antibiotic resistance cassette.…”

Section: Strain Constructionsmentioning

confidence: 99%

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

et al. 2016

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The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180°on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus.

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“…For visual clarity, allele numbers and polypeptide designations are expressed as superscripts when more than one recBCD gene or RecBCD polypeptide are designated. Bacterial strains were constructed by phage P1 transduction, CaCl 2 -mediated transformation, electroporation, or "recombineering" (Ausubel et al 2003;Thomason et al 2005). Plasmids were constructed by standard procedures (Ausubel et al 2003).…”

Section: Bacterial Strains Phage and Plasmidsmentioning

confidence: 99%

Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots

Amundsen¹,

Taylor²,

Reddy³

et al. 2007

Genes Dev.

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The Escherichia coli RecBCD helicase-nuclease, a paradigm of complex protein machines, initiates homologous genetic recombination and the repair of broken DNA. Starting at a duplex end, RecBCD unwinds DNA with its fast RecD helicase and slower RecB helicase on complementary strands. Upon encountering a Chi hot spot (5-GCTGGTGG-3), the enzyme produces a new 3 single-strand end and loads RecA protein onto it, but how Chi regulates RecBCD is unknown. We report a new class of mutant RecBCD enzymes that cut DNA at novel positions that depend on the DNA substrate length and that are strictly correlated with the RecB:RecD helicase rates. We conclude that in the mutant enzymes when RecD reaches the DNA end, it signals RecB's nuclease domain to cut the DNA. As predicted by this interpretation, the mutant enzymes cut closer to the entry point on DNA when unwinding is blocked by another RecBCD molecule traveling in the opposite direction. Furthermore, when RecD is slowed by a mutation altering its ATPase site such that RecB reaches the DNA end before RecD does, the length-dependent cuts are abolished. These observations lead us to hypothesize that, in wild-type RecBCD enzyme, Chi is recognized by RecC, which then signals RecD to stop, which in turn signals RecB to cut the DNA and load RecA. We discuss support for this "signal cascade" hypothesis and tests of it. Intersubunit signaling may regulate other complex protein machines.[Keywords: Homologous recombination; E. coli; RecBCD enzyme; Chi sites; complex protein machines] Supplemental material is available at http://www.genesdev.org.

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Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Cited by 61 publications

References 12 publications

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Colocalization of distant chromosomal loci in space in E. coli: a bacterial nucleolus

Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots

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