Genetic Recombination of Bacteriophage T7 Dna in Vitro

Sadowski, Paul D.; Bradley, William A.; Lee, Donald; Roberts, Larry E.

doi:10.1016/b978-0-12-048850-6.50085-5

Cited by 17 publications

(17 citation statements)

References 25 publications

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“…However, biochemical studies support a more direct role of 17 gene 2.5 protein in recombination. Upon infection of E. coli with 17 phage, a single-stranded DNA renaturation activity is induced, suggesting that 17 gene 2.5 protein is involved in this activity (17). It has, in fact, recently been shown that gene 2.5 protein facilitates the renaturation of single-stranded DNA much more efficiently that does E. coli SSB, recA, or T4 gene 32 protein (S. Tabor and C.C.R., unpublished results).…”

Section: Discussionmentioning

confidence: 93%

Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication.

Kim

Richardson

1993

Proc. Natl. Acad. Sci. U.S.A.

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Section: Discussionmentioning

confidence: 93%

Bacteriophage T7 gene 2.5 protein: an essential protein for DNA replication.

Kim

Richardson

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently it has been found that T7 single-strand-DNA binding protein and the helicase encoded by gene 4 play major roles in annealing complementary DNA strands together (16,19,20). Thus, formation of single-stranded DNA and annealing of those strands to form heteroduplexes may figure prominently in T7 recombination (42). The uncertainty regarding the recombination process in T7 underscores the need for more information regarding what happens at a double-strand break in T7 that causes increased recombination and increased deletion when the break forms between a pair of direct repeats.…”

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confidence: 99%

In Vitro Repair of Gaps in Bacteriophage T7 DNA

Lai

Masker

1998

J Bacteriol

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An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.

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“…First, we used favorable conditions, namely, enriched medium and a high MOI to favor adsorption. Studier (21) noted adsorption problems encountered in the past, and he and others (3,19) used a high MOI (.10) when working with phage T7. The use of a relatively low MOI for multicomplex formation may have prevented Schweiger et al (20) from demonstrating MR in UV-irradiated phage T7.…”

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confidence: 99%