Sequence of the bacteriophage P22 Anti-RecBCD (abc) genes and properties of P22 abc region deletion mutants

Murphy, Kenan C.; Fenton, Anita C.; Poteete, Anthony R.

doi:10.1016/0042-6822(87)90017-1

Cited by 28 publications

(21 citation statements)

References 28 publications

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“…In the absence of plasmid-borne phage functions, crossing-over in the RFLP substitution occurred at a high rate: 14% of the hybridizing DNA extracted from infected cells was in the 5,400-bp recombinant band. Supplying the products of P22 genes abcl, abc2, and a third gene of unknown function called orf-56 (22) from plasmid pTP233 reduced recombination eightfold; supplying Gam from plasmid pTP224 had a similar but smaller effect, reducing production of the 5,400-bp fragment to 5.2%. In this experiment, Gam had an additional effect, stimulating phage replication eightfold.…”

Section: Resultsmentioning

confidence: 94%

“…The construction of P22 strains bearing the deletions A183, A327, and A328 has been described (13,22). These deletions were combined with the ampicillin resistance-conferring immI substitution Ap3l pfrl (40) by crossing with P22 Ap3l pfrl.…”

Section: Plasmidmentioning

confidence: 99%

“…Two adjacent genes, abcl and abc2, contribute independently to this function; abc2 makes the larger contribution (22). The abc2 gene, in particular, is analogous to the X gam gene in that it is required for growth of the phage in a polA host.…”

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confidence: 99%

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Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions

1988

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Plasmids that express the bacteriophage A gam gene or the P22 abc2 gene (with and without abcl) at controllable levels were placed in Escherichia coli and tested for effects on the activity of RecBCD. Like Gam, Abc2 inhibited the ATP-dependent exonuclease activity of RecBCD, apparently not by binding to DNA. However, Abc2-mediated inhibition was partial, while Gam-mediated inhibition was complete. Both Abc2 and Gam inhibited host system-mediated homologous recombination in a Chi-containing interval in the chromosome of a hybrid A phage; Abc2 inhibited it more strongly than Gam. Gam but not Abc2 spared a phage T4 gene 2 mutant from restriction by RecBCD; Abc2 exhibited weak sparing activity in combination with Abcl and substantial activity in combination with both Abcl and the P22 homologous recombination function Erf. Either Gam or the combination of the A recombination functions Exo and Bet was sufficient to induce a mode of plasmid replication that produced linear multimers. The combination of Abc2, Abcl, and Erf also exhibited this activity. However, Erf was inactive, both by itself and in combination with Abcl; Abc2 had weak activity. These results indicate that Gam and Abc2 modulate the activity of RecBCD in significantly different ways. In comparison with A Gam, P22 Abc2 has a weak effect on RecBCD nuclease activity but a strong effect on its recombination-promoting activity.The RecBCD enzyme of Escherichia coli catalyzes exonucleolytic degradation of DNA and promotes homologous recombination. To a certain extent, these two activities are distinct; while recB and recC mutants lack both, recD mutants lack only the nuclease (1, 6). The presence of RecBCD nuclease in the cell inhibits rolling-circle replication of such diverse replicons as phage X and plasmid ColEl, possibly because a critical part of the rolling circle is susceptible to exonucleolytic degradation (9,12,35). Accordingly, many bacteriophages, including X, T2, T3, T4, T5, T6, T7, P1, and 480, inhibit RecBCD nuclease during lytic growth (3, 24, 28). In addition to inhibiting rolling-circle replication, the RecBCD nuclease can degrade the linear DNA injected by some phages. Phage T4 virions contain a protein, the product of phage gene 2, that protects the phage chromosome from RecBCD; phage mutants lacking this function are restricted to growth in recBCD mutants (23 of RecBCD on lytic growth of the phage (13). Two adjacent genes, abcl and abc2, contribute independently to this function; abc2 makes the larger contribution (22). The abc2 gene, in particular, is analogous to the X gam gene in that it is required for growth of the phage in a polA host. However, the gam and abc genes differ in significant ways. They are not homologous. Moreover, they occupy different positions in the phage chromosome: the abc genes are located to the left of the homologous recombination-promoting erf gene, while gam is located to the right of the homologous recombination-promoting bet gene. This is the only known case in which the map order of analogous genetic elemen...

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Section: Resultsmentioning

confidence: 94%

Section: Plasmidmentioning

confidence: 99%

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Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions

1988

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“…It may be this (or a similar) activity which remains intact in Gam+ hosts and accounts for the wild-type levels of conjugational recombination seen in the presence of Gam. Genetic analyses have also linked RecBCD to DNA polymerase I. recBC polA double mutants are lethal (27), phage devoid of their RecBCD-modifying functions (A gam and P22 abc) fail to grow on polA strains (38,67), and both recBC and polA are involved in the repair of double-stranded breaks after exposure to UV light (52). The significance of these observations with regard to a possible physical link of RecBCD with DNA polymerase I has yet to be determined.…”

Section: Discussionmentioning

confidence: 99%

Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme

1991

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The Gam protein was isolated from cells containing a Gam-producing plasmid. The purified Gam protein was found to bind to RecBCD without displacing any of its subunits. Gam was shown to inhibit all known enzymatic activities of RecBCD: ATP-dependent single-and double-stranded DNA exonucleases, ATP-independent single-stranded endonuclease, and the ATP-dependent helicase. When produced in vivo, Gam inhibited X-activated recombination in A red gam crosses but had little effect on the host's ability to act as a recipient in conjugational recombination. These experiments suggest that RecBCD possesses an additional "unknown" activity that is resistant to or induced by Gam. Additionally, the expression of Gam in recD mutants sensitizes the host to UV irradiation, indicating that Gam alters one or more of the in vivo activities of RecBC(D-).The primary route of general homologous recombination in Escherichia coli proceeds through the RecBCD pathway. The RecBCD enzyme (also known as ExoV) has five known activities: ATP-dependent double-stranded and singlestranded DNA (dsDNA and ssDNA, respectively) exonucleases, an ATP-stimulated ssDNA endonuclease, an ATPdependent unwinding activity, and a DNA-dependent ATPase (for reviews, see references 14, 53, 58, and 62). The enzyme has also been shown to recognize and promote nicking at X sites (5'GCTGGTGG3') while unwinding ds DNA in vitro (42). At one time or another, one or more of these activities have been implicated as the key function(s) involved in the enzyme's ability to promote homologous recombination (1-4, 10-12, 21, 27, 28, 32, 41, 50, 59). Some of these studies involved the correlation of residual activities of RecBCD enzyme isolated from mutants carrying recB, recC, or recD with the cell's ability to carry out recombination and repair.The approach taken in this report is similar to those described above: an examination of the residual biochemical and genetic activities of RecBCD after modification. However, the modification employed here is afforded not by mutation but by the A Gam protein, which has been reported to bind RecBCD and inhibit the exonuclease and ATPase activities (25). The unwinding and X-recognizing capabilities of RecBCD were not known at the time of earlier studies and thus were not tested. It has been a general assumption since those studies that a cell containing Gami was effectively a recBC null mutant. However, Hays et al. (22) and others (44) showed that Gam-producing plasmids do not confer a recBC phenotype on the host with regard to recombination following conjugation, even though such hosts display other recBC phenotypes such as UV sensitivity, the ability to plate T4 2-phage, inhibition of X recombination in a X-containing interval, and decreased cell viability (19,44 (2,19,31,44).Despite these similarities, there are distinct differences between Gam-containing hosts and recD mutants with regard to which activities of RecBC are retained and how the modification affects x-stimulated X recombination. This report describes a RecBCD-Gam complex...

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“…Phage Abc function (an analog of the lambda Gam function) inhibits the exonuclease activity of RecBCD enzyme and the ability to promote homologous recombination (assayed by conjugation and phage lambda crosses) (44,49). By inhibiting the RecBCD exonuclease, Abc protects the short terminally redundant ends of the phage genome prior to circularization and protects the end of the concatemeric genome required for packaging (43). The phage Erf protein (an analog of lambda ␤ protein) promotes pairing of homologous single strands (48), which contributes to the ability of P22 to transduce plasmids (18) and promotes recombination in phage lambda and phage P22 crosses (50).…”

Section: Vol 178 1996mentioning

confidence: 99%

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination

Miesel

Roth

1996

J Bacteriol

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A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (ϳ3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10-to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBCdependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.

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Sequence of the bacteriophage P22 Anti-RecBCD (abc) genes and properties of P22 abc region deletion mutants

Cited by 28 publications

References 28 publications

Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions

Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions

Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination

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