λ Rap protein is a structure-specific endonuclease involved in phage recombination

Sharples, Gary J.; Corbett, Lisa; Graham, Ian R.

doi:10.1073/pnas.95.23.13507

Cited by 28 publications

(39 citation statements)

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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: 81%

“…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). We tested whether nin genes could functionally replace any of the E. coli genes needed for Red-mediated gene replacement, by inserting a P tac fusion of orf and other genes downstream of it into the galK gene.…”

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confidence: 99%

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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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confidence: 81%

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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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“…4b) could also belong to this family. rap encodes a structure-specific endonuclease that is involved in phage recombination (Sharples et al, 1998). Located in the nin non-essential region of lambdoid phages, it seems to be a (nonhomologous) alternative to rusA-like genes, also encoding structure-specific endonucleases (Hendrix et al, 1999).…”

Section: Dna Endonucleasesmentioning

confidence: 99%

Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are bIL170, AF009630; bIL120, AY054975; bIL15, AY054976; bIL191, AY054977; bIL77, AY054978.

et al. 2002

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The complete 31 754 bp genome of bIL170, a virulent bacteriophage of Lactococcus lactis belonging to the 936 group, was analysed. Sixty-four ORFs were predicted and the function of 16 of them was assigned by significant homology to proteins in databases. Three putative homing endonucleases of the HNH family were found in the early region. An HNH endonuclease with zinc-binding motif was identified in the late cluster, potentially being part of the same functional module as terminase. Three putative structural proteins were analysed in detail and show interesting features among dairy phages. Notably, gpl12 (putative fibre) and gpl20 (putative baseplate protein) of bIL170 are related by at least one of their domains to a number of multidomain proteins encoded by lactococcal or streptococcal phages. A 110-to 150-aa-long hypervariable domain flanked by two conserved motifs of about 20 aa was identified. The analysis presented here supports the participation of some of these proteins in host-range determination and suggests that specific adsorption to the host may involve a complex multi-component system. Divergences in the genome of phages of the 936 group, that may have important biological properties, were noted. Insertions/deletions of units of one or two ORFs were the main source of divergence in the early clusters of the two entirely sequenced phages, bIL170 and sk1. An exchange of fragments probably affected the regions containing the putative origin of replication. It led to the absence in bIL170 of the direct repeats recognized in sk1 and to the presence of different ORFs in the ori region. Shuffling of protein domains affected the endolysin (putative cell-wall binding part), as well as gpl12 and gpl20.

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“…The RusA protein is a Holliday junction resolvase, which can substitute for the RuvC protein of E. coli in promoting recombination (7,15). Consistent with this proposal, it was found that the rap gene encodes a nuclease which cleaves at the branch points of Holliday junctions; in addition, it cleaves three-stranded junctions (D-loops) (16).…”

mentioning

confidence: 61%

“…The properties of rap ϩ cells suggest that Rap can substitute for RuvC in some cellular pathways but not in others. On the other hand, the Rap protein's partial complementation of a recF mutant (Table 2) and its broader range of nucleolytic activities (16) suggests that, conversely, Rap may carry out an additional function(s), most likely in phage replication, repair, or recombination, that RuvC does not.…”

Section: Strainsmentioning

confidence: 99%

Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

2002

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The rap gene of bacteriophage was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recG⌬ red ؉ rap ؉ strain 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. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicolresistant bacterial progeny. The results of these experiments indicated that the rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.

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λ Rap protein is a structure-specific endonuclease involved in phage recombination

Cited by 28 publications

References 29 publications

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

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

Recombination-Promoting Activity of the Bacteriophage λ Rap Protein in Escherichia coli K-12

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