The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E. coli

Sawitzke, James A.; Stahl, Franklin W.

doi:10.1128/jb.176.21.6730-6737.1994

Cited by 34 publications

(26 citation statements)

References 49 publications

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“…These findings are superficially inconsistent with those of a previous study, which suggested a more limited capability of Orf to substitute for RecORF (34). However, in the previous study, orf was expressed from a plasmid.…”

Section: Discussioncontrasting

confidence: 56%

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Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Poteete

2004

J Bacteriol

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The orf gene of bacteriophage , fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a ⌬(recC ptr recB recD)::P tac gam bet exo pae cI ⌬recG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.Conjugative recombination in a recBC sbcB sbcC mutant of Escherichia coli depends upon the recF, recO, and recR genes, as well as other rec genes and the ruv genes, and is said to proceed via the RecF pathway (for a review, see reference 14). In the same cell, however, homologous recombination between bacteriophage chromosomes does not depend on recF, recO, or recR, even when the phage lacks its own recombination system, Red, and is thus dependent upon the host's recombination system (33). Sawitzke and Stahl (33) showed that 's RecORF independence is due to its orf gene (previously called ninB). Further studies showed that orf encodes a protein and that plasmid-borne orf can act in trans to promote phage recombination, but not conjugative recombination, in the absence of RecORF (34).Recombination between phage chromosomes via the Red pathway requires neither RecORF nor Orf, but Orf nonetheless is a participant (39). The result of its action is to focus crossovers near the initiating double-strand break in both the RecF (35) and Red (39) pathways.An additional observation suggested that Orf can promote recombination events other than crossovers between phage chromosomes. In a cell in which the recC-ptr-recB-recD gene cluster is replaced by the Red system genes (gam, bet, and exo) and from which the recG gene is deleted, recombination between the bacterial chromosome and a 3.6-kbp linear double-stranded DNA (dsDNA) molecule is dependent upon recF, recO, and recR. However, in such a cell, ectopic expression from the bacterial chromosome of a segment of the chromosome, including the orf gene, partially decreases this dependence (29).For this study, the orf gene was installed by itself in the galK locus. The expression of chromosomally encoded orf was found to have surprisingly pleiotropic effects on recombination, replication, and repair in E. coli. Orf suppressed the mutant phenotypes of not only recF, recO, and recR, but ruvAB and ruvC as well. In contrast, the recA803 allele, which partially suppresses the phenotypes of recF, recO, and recR mutations affecting RecF pathway recombination and repair (44, 45), had no comparable effect on the Red pathway. MATERIALS AND METHODSBacterial strains. The bacterial strains used for this study are described in Table 1. Some of the constructions involved the repla...

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Section: Discussioncontrasting

confidence: 56%

“…Orf is deleterious to E. coli, as previously noted by Sawitzke and Stahl (34). Expression of the orf gene from a multicopy plasmid (a pBR322 derivative) directed by the moderate, lac repressor-controllable promoter P mac was found to be lethal to wild-type E. coli, even in a strain bearing FЈ lacI Q .…”

Section: Resultsmentioning

confidence: 75%

Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Poteete

2004

J Bacteriol

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“…Results presented above do not distinguish which proteins encoded by nin genes contribute to complementation of the recombination defects of mutant strains lacking RecFOR or RuvC. However, research by others on orf and rap strongly suggests that these are the relevant functions (11,26,28). The mechanism shown in Fig.…”

mentioning

confidence: 96%

“…Two genes in the nin region of phage , orf and rap, function in homologous recombination. The orf gene can substitute for recF, recO, and recR in phage recombination mediated by the host system in recBC sbcBC cells in the absence of the Red system (26). The rap gene encodes an endonuclease which specifically cleaves branched DNA structures, including Holliday junctions, that are thought to be generated during recombination and which hypothetically might substitute for RuvC (11,28).…”

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confidence: 99%

Genetic Requirements of Phage λ Red-Mediated Gene Replacement in Escherichia coli K-12

Poteete

Fenton

2000

J Bacteriol

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Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac ؊ chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.Efficient recombination involving the Escherichia coli chromosome takes place only when the recombining partner DNA is large and contains Chi sites to activate the recombinationpromoting activities of RecBCD (for a review, see reference 20). Short linear DNA molecules in the cell generally are destroyed by RecBCD. Recombination between short linear DNA molecules and the host chromosome at a frequency high enough to be of practical use in making gene replacements has been observed with recD and recBC sbcBC mutant strains (12, 24). Still-higher frequency recombination is seen with E. coli strains in which the recBCD gene cluster is replaced by the red genes (gam, bet, and exo) of phage (19).In addition to proceeding at high efficiency, Red-mediated recombination between a short linear DNA molecule and a circular homologue may represent a simpler recombination pathway than any of the previously characterized pathways for conjugational or transductional recombination. These properties of efficiency and (relative) simplicity recommend the hybrid phage-bacterial recombination system for research on general recombination mechanisms. In previous studies, we have shown that such recombination events require the activities of RecA, Exo, and Bet, as well as double-strand breaks (22). Murphy (19) found that the frequency is decreased by mutation of recA and recF and increased by mutation of recJ. The frequency of Red-mediated recombination is also elevated in a recG mutant strain. In the case of an event involving the insertion of substantial nonhomology (as in gene replacement), recombination in the recG host is apparently constrained to proceed through a pathway requiring RuvC resolvase (23).In this study, we examined the dependence of Red-mediated gene replacement on several additional known E. coli recombination genes. Functional complementation between some of these genes and other phage genes was tested as well.Strains. lac::cat819 nin5 has been described previously (23)....

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“…In vitro, the same RecA441 (previously tif; E38K, I298V) and RecA803 (V37M) proteins exhibit an enhanced capacity to displace SSB and bind ssDNA (40,41). In addition, a gene in bacteriophage called ninB or orf (described further below) has been identified that can replace recF, recO, and recR functions in lambda recombination (42,43). In vivo, mutant bacteria missing any of the recFOR functions exhibit a delayed activation of the SOS response that might reflect slow formation of the RecA filaments required to facilitate LexA cleavage (44,45).…”

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confidence: 99%

SSB Protein Limits RecOR Binding onto Single-stranded DNA

Hobbs¹,

Sakai

Cox

2007

Journal of Biological Chemistry

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The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB proteincoated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSBcoated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

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The phage lambda orf gene encodes a trans-acting factor that suppresses Escherichia coli recO, recR, and recF mutations for recombination of lambda but not of E. coli

Cited by 34 publications

References 49 publications

Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Modulation of DNA Repair and Recombination by the Bacteriophage λ Orf Function inEscherichia coliK-12

Genetic Requirements of Phage λ Red-Mediated Gene Replacement in Escherichia coli K-12

SSB Protein Limits RecOR Binding onto Single-stranded DNA

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