Roles for λ Orf and Escherichia coli RecO, RecR and RecF in λ Recombination

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 357-369
Author(s):  
James A Sawitzke ◽  
Franklin W Stahl
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Gene Product ◽  
Strand Break ◽  
Bacteriophage Λ ◽  
Recf Pathway ◽  
The Right ◽  
Mutant Cells ◽  
Λ Red ◽  
Red Recombination

Bacteriophage λ lacking its Red recombination functions requires either its own gene product, Orf, or the product of Escherichia coli's recO, recR and recF genes (RecORF) for efficient recombination in recBC sbcB sbcC mutant cells (the RecF pathway). Phage crosses under conditions of a partial block to DNA replication have revealed the following: (1) In the presence of Orf, RecF pathway recombination is similar to λ Red recombination; (2) Orf is necessary for focusing recombination toward the right end of the chromosome as λ is conventionally drawn; (3) RecORF-mediated RecF pathway recombination is not focused toward the right end of the chromosome, which may indicate that RecORF travels along the DNA; (4) both Orf- and RecORF-mediated RecF pathway recombination are stimulated by DNA replication; and (5) low level recombination in the simultaneous absence of Orf and RecORF may occur by a break-copy mechanism that is not initiated by a double strand break. Models for the roles of Orf and RecO, RecR and RecF in recombination are presented.

scholarly journals Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

Virology ◽  
2003 ◽  
Vol 313 (2) ◽  
pp. 622-628 ◽  
Author(s):  
Barbara Kędzierska ◽  
Monika Glinkowska ◽  
Adam Iwanicki ◽  
Michał Obuchowski ◽  
Piotr Sojka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Host Cell ◽  
Gene Product ◽  
Bacteriophage Λ

scholarly journals The ftsA Gene Product: a Possible Connection between DNA Replication and Septation in Escherichia coli

Microbiology ◽  
1985 ◽  
Vol 131 (2) ◽  
pp. 239-244
Author(s):  
A. TORMO ◽  
C. FERNANDEZ-CABRERA ◽  
M. VICENTE
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Gene Product

scholarly journals High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

2014 ◽  
Vol 80 (13) ◽  
pp. 3826-3834 ◽  
Author(s):  
Junjie Yang ◽  
Bingbing Sun ◽  
He Huang ◽  
Yu Jiang ◽  
Liuyang Diao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Modification ◽  
Target Gene ◽  
Donor Plasmid ◽  
Content Type ◽  
Recognition Sites ◽  
Genetic Modifications ◽  
Pcr Targeting ◽  
Λ Red ◽  
Red Recombination

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

scholarly journals Heteroduplex chain polarity in recombination of phage lambda by the red, RecBCD, RecBC(D-) and RecF pathways.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 7-22 ◽  
Author(s):  
I Siddiqi ◽  
M M Stahl ◽  
F W Stahl
Keyword(s):  
Escherichia Coli ◽  
Genetic Map ◽  
Bacteriophage Lambda ◽  
The Other ◽  
Genetic Contribution ◽  
Phage Lambda ◽  
Heteroduplex Dna ◽  
Recf Pathway ◽  
The Right ◽  
Parental Information

Abstract We have examined the chain polarity of heteroduplex DNA in unreplicated, bacteriophage lambda splice recombinants when recombination was by the RecBCD, RecBC(D-), or RecF pathway of Escherichia coli or the Red pathway of lambda. For each of these pathways, recombination is activated by the cutting of cos that accompanies chromosome packaging, and is effected by recombination enzymes acting at the right end created by that cutting. For exchanges occurring near cos, one parent makes a lesser physical and genetic contribution than does the other. For each pathway, when the phage carried standard cos, this minority contribution was predominantly on the r chain, ending 5' at the right end of lambda. When standard cos was replaced by a cloned inverted cos located centrally on the standard lambda genetic map, minority contribution was predominantly on the l chain. In each case, the polarity of the overlap was usually that formed by 3' overhangs of parental information and material. These results are discussed in the context of current models of recombination for the different pathways.

Recombination of bacteriophage λ in recD mutants of Escherichia coli

Genome ◽  
1989 ◽  
Vol 31 (1) ◽  
pp. 53-67 ◽  
Author(s):  
David S. Thaler ◽  
Elizabeth Sampson ◽  
Imran Siddiqi ◽  
Susan M. Rosenberg ◽  
Lynn C. Thomason ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Restriction Enzyme ◽  
Null Alleles ◽  
Mutant Strains ◽  
Recbcd Enzyme ◽  
Mutant Cells ◽  
D Subunit

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red−Gam−phage λ is strikingly altered by recD null mutations in the host. When λ DNA replication is blocked, recombination in recD mutant strains is high near λ's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along λ unless the λ chromosome contains a χ sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on λ. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent χ). The distribution of exchanges in recD mutants is strongly affected by λ DNA replication. The distribution of exchanges on λ growing in rec+ cells is not influenced by DNA replication. The exchange distribution along λ in recD mutant cells is independent of χ in a variety of conditions. Recombination in rec+ cells is χ influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for λ recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break–repair, as it does in λ's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a χ sequence.Key words: χ sequence, RecBCD pathway, Red pathway, RecBC‡ pathway.

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