Protection of parental T4 DNA from a restriction exonuclease by the product of gene 2

Oliver, Donald B.; Goldberg, Edward B.

doi:10.1016/0022-2836(77)90276-5

Cited by 87 publications

(62 citation statements)

References 8 publications

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“…Two assays indicated that the mutants retained RecBCD exonuclease activity. This activity degrades the DNA of phage T4 lacking the gene 2 protein, which is thought to bind to the ends of the linear DNA in the virion and thereby protect the DNA from RecBCD exonuclease upon injection into an E. coli cell (Oliver and Goldberg 1977). T4 gene 2 mutant phage formed plaques with the same low efficiency (∼10 −6 ) on the new mutants as on recBCD + cells (Table 1) but formed plaques with near unit efficiency on previously isolated mutants lacking RecBCD exonuclease (⌬recBCD or ⌬recD).…”

Section: Isolation Of a Novel Class Of Rec − Nuc + Recbcd Mutantsmentioning

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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Section: Isolation Of a Novel Class Of Rec − Nuc + Recbcd Mutantsmentioning

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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“…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).…”

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confidence: 99%

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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“…One model is that a very modest amount of MR-dependent RDR is necessary to generate concatameric DNA suitable for DNA packaging, and the MR-depleted infection can achieve this level of RDR. Another model involves the ends of the infecting T4 chromosome, which are bound by the end-protection protein gp2 (Silverstein and Goldberg 1976a,b;Oliver and Goldberg 1977;Lipinska et al 1989). Eukaryotic MR complex is needed to liberate DNA from SPO11-DNA covalent complexes by means of an endonuclease reaction near the bound protein (for review, see Paull 2010).…”

Section: Discussionmentioning

confidence: 99%

Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

et al. 2013

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The in vivo functions of the bacteriophage T4 Mre11/Rad50 (MR) complex (gp46/47) in double-strand-end processing, double-strand break repair, and recombination-dependent replication were investigated. The complex is essential for T4 growth, but we wanted to investigate the in vivo function during productive infections. We therefore generated a suppressed triple amber mutant in the Rad50 subunit to substantially reduce the level of complex and thereby reduce phage growth. Growth-limiting amounts of the complex caused a concordant decrease in phage genomic recombination-dependent replication. However, the efficiencies of doublestrand break repair and of plasmid-based recombination-dependent replication remained relatively normal. Genetic analyses of linked markers indicated that double-strand ends were less protected from nuclease erosion in the depleted infection and also that end coordination during repair was compromised. We discuss models for why phage genomic recombination-dependent replication is more dependent on Mre11/Rad50 levels when compared to plasmid recombination-dependent replication. We also tested the importance of the conserved histidine residue in nuclease motif I of the T4 Mre11 protein. Substitution with multiple different amino acids (including serine) failed to support phage growth, completely blocked plasmid recombination-dependent replication, and led to the stabilization of double-strand ends. We also constructed and expressed an Mre11 mutant protein with the conserved histidine changed to serine. The mutant protein was found to be completely defective for nuclease activities, but retained the ability to bind the Rad50 subunit and double-stranded DNA. These results indicate that the nuclease activity of Mre11 is critical for phage growth and recombination-dependent replication during T4 infections.

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Protection of parental T4 DNA from a restriction exonuclease by the product of gene 2

Cited by 87 publications

References 8 publications

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

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

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

Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

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