Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Roberts, Larry E.; Sadowski, Paul D.; Wong, J. Tze‐Fei

doi:10.1021/bi00266a042

Cited by 4 publications

(5 citation statements)

References 26 publications

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“…(47) that showed that the first cleavage made by the exonuclease activity of gp6 at the 5Ј-terminus is not stimulated by gp2.5 because the substrate used in that study did not have an adjacent ssDNA region (47).…”

Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 90%

“…The finding that E. coli ssDNA protein did not stimulate gp6 implies that there is an interaction of gp2.5 with gp6. However, no physical interaction between the two proteins was detected (47).…”

Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 96%

“…gp2.5 not only removes secondary structure in ssDNA; it also physically interacts with other T7 replication proteins to coordinate the multiple reactions occurring at the replication fork (7,36). Previous studies found that gp2.5 stimulates the exonuclease activity of gp6 on double-stranded DNA, particularly in the presence of 100 mM NaCl, a salt concentration that inhibits gp6 activity (47). The finding that E. coli ssDNA protein did not stimulate gp6 implies that there is an interaction of gp2.5 with gp6.…”

Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 99%

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Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Mitsunobu

Zhu

Lee

et al. 2014

Journal of Biological Chemistry

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Background: Flap endonucleases remove 5Ј-single-stranded DNA termini. Results: T7 gene 6 exonuclease is a flap endonuclease that cleaves 5Ј-single-stranded termini one nucleotide into the duplex. Conclusion:The flap endonuclease activity catalyzes the removal 5Ј-single-stranded tails arising from duplex DNA. Significance: The specificity of gene 6 protein identifies it as a flap endonuclease that can remove unusual structures of recombination and replication.

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Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 90%

Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 96%

Section: Stimulation Of Gp6 Activity By T7 Gene 25 Ssdna-bindingmentioning

confidence: 99%

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Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Mitsunobu

Zhu

Lee

et al. 2014

Journal of Biological Chemistry

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“…In other biological systems, a critical role of single strand DNA binding protein is to protect exposed single-stranded DNA from nuclease attack (2). In T7 the single strand binding protein enhances the activity of gene 6 exonuclease so that apparent nuclease degradation is actually more pronounced with the gene 2.5 protein than without it (46). Thus, available data provide no evidence to suggest that excessive DNA degradation causes the inhibition of recombinational repair associated with gene 2.5 inactivation.…”

Section: Discussionmentioning

confidence: 99%

T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair

Yu¹,

Masker

2001

J Bacteriol

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An in vitro system based on Escherichia coli infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a unique XhoI site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected E. coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an E. coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E. coli recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.Double strand breaks are a frequent hazard to DNA molecules. These breaks can result from a number of causes, including DNA damage, fractures at progressing replication forks, or as part of normal homologous recombination (10,15,26,37,61). Failure to repair a double strand break can be lethal. Improper repair of double strand breaks can lead to deletions or chromosome aberrations (9, 12, 52). To minimize the deleterious effects of double strand breaks, living organisms maintain an array of repair mechanisms able to correct double strand breaks or rescue partial genomes formed as a result of breaks (3,10,12,21,22,26,38,39). Recombinationinduced DNA replication is a major route to rescue of partial genomes formed by collapse of replication forks in prokaryotes (26). In Escherichia coli, stable DNA replication is induced by forming new replication forks at sites other than the normal origin of replication (22,27,32). In bacteriophage T4, recombination between partial genomes formed by double strand breaks and intact T4 DNA molecules induces new replication forks that comprise a major part of the phage's DNA replication cycle (25, 36). It has long been appreciated that reco...

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“…Thus, the kinetics of ligation should be a measure of the extent of DNA looping. The gene 6 exonuclease of phage T7 hydrolyzes double-stranded DNA in a 5 0 to 3 0 direction (Roberts et al, 1982). A fixed amount of labeled DNA either by itself or after preincubation with just the monomers, or first preincubation with monomers followed by the dimers, was treated for the indicated time periods with DNA ligase at 12ºC.…”

Section: Optimal Incc-orif Looping Required the Presence Of Both Monomeric And Dimeric Repementioning

confidence: 99%

Oligomeric Initiator Protein-Mediated DNA Looping Negatively Regulates Plasmid Replication In Vitro by Preventing Origin Melting

Zzaman

Bastia

2005

Molecular Cell

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Although DNA looping between the initiator binding sites (iterons) of the replication origin (ori) of a plasmid and the iterons located in a cis-acting control sequence called inc has been postulated to promote negative control of plasmid DNA replication, not only was definitive evidence for such looping lacking, but also the detailed molecular mechanism of this control had not been elucidated. Here, we present direct evidence showing that both the monomeric and the dimeric forms of the RepE initiator protein of F factor together promote pairing of incC-oriF sites by DNA looping. By using a reconstituted replication system consisting of 26 purified proteins, we show further that the DNA loop formation negatively regulates plasmid replication by inhibiting the formation of an open complex at the replication origin, thus elucidating a key step of replication control.

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Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Cited by 4 publications

References 26 publications

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair

Oligomeric Initiator Protein-Mediated DNA Looping Negatively Regulates Plasmid Replication In Vitro by Preventing Origin Melting

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