Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

Lai, Ying-Ta; Masker, Warren E.

doi:10.1128/jb.182.2.327-336.2000

Cited by 4 publications

(18 citation statements)

References 59 publications

(75 reference statements)

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“…In contrast to what is found with E. coli and with bacteriophage T4 (11,22,25,36), T7 double strand break repair is not associated with extensive DNA replication (28). This argues that either re-formation of new replication forks at break sites is not common in T7 or that alternative repair mechanisms are sufficiently robust to allow a high level of repair even when replicative repair mechanisms cannot operate (28).…”

contrasting

confidence: 47%

“…Thus, a double strand break will eliminate the 3.6-kb KpnI D fragment and leave instead fragments that are 2.6 and 1.0 kb long. Repair of the double strand break restores the integrity of the KpnI D fragment (28). Experiments like that displayed in Table 1, which used BstXI fragments as donor DNA, proved difficult to interpret because the unused donor DNA was not extensively digested during the course of the experiment and some of the residual BstXI bands run very near the position expected for the KpnI D band.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction mixtures were extracted with phenol-chloroform and precipitated with ethanol, and the DNA was resuspended in 1/10 volume Tris-EDTA (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA). The DNA was treated with boiled bovine pancreas RNase a (final concentration, 1 mg/ml, Calbiochem) and digested with KpnI, and 0.014 ml was subjected to electrophoresis at 24 V for 4 h in a 0.4% agarose gel in Tris acetate-EDTA buffer (28,29). The gel were stained with ethidium bromide to give the patterns shown in the figures.…”

Section: Methodsmentioning

confidence: 99%

“…When 1.5 nmol of BstXI-digested donor DNA was incubated with 1.5 nmol of T7 genomes, each with a double strand break, there is one molecule of the relevant restriction fragment for each double strand break. The 2,136-bp PCR fragment extending from positions 5594 to 7729, which served as donor DNA for some experiments, was prepared as previously described (28). In experiments using the PCR fragment as donor DNA, the number of PCR fragments in a repair reaction was 10 times the number of T7 genomes.…”

Section: Methodsmentioning

confidence: 99%

“…This argues that either re-formation of new replication forks at break sites is not common in T7 or that alternative repair mechanisms are sufficiently robust to allow a high level of repair even when replicative repair mechanisms cannot operate (28).…”

mentioning

confidence: 99%

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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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confidence: 47%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein

Hernandez

Richardson

2019

Seminars in Cell & Developmental Biology

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The essential bacteriophage T7-encoded single-stranded DNA binding protein is the nexus of T7 DNA metabolism. Multiple layers of macromolecular interactions mediate its function in replication, recombination, repair, and the maturation of viral genomes. In addition to binding ssDNA, the protein binds to DNA polymerase and DNA helicase, regulating their activities. The protein displays potent homologous DNA annealing activity, underscoring its role in recombination.

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Repair of double‐strand breaks by incorporation of a molecule of homologous DNA

Lai

Masker²

2000

Molecular Microbiology

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SummaryAn in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules (`donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending < 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32 P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [ 32 P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.

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Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

Cited by 4 publications

References 59 publications

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

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

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein

Repair of double‐strand breaks by incorporation of a molecule of homologous DNA

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