Studies on bacteriophage T7 DNA synthesis in vitro

ABSTRACT An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5′→3′ exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.

Download Full-text

Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)43926-4 ◽

1980 ◽

Vol 255 (16) ◽

pp. 7956-7964

Author(s):

H. Fischer ◽

D.C. Hinkle

Keyword(s):

Dna Replication ◽

Rna Polymerase ◽

Dna Synthesis ◽

T7 Rna Polymerase ◽

Bacteriophage T7 ◽

Stimulation Of

Download Full-text

In vitro concatemerization of bacteriophage T7 DNA: Role of DNA synthesis and gene 6 exonuclease

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o85-034 ◽

1985 ◽

Vol 63 (4) ◽

pp. 237-242 ◽

Cited By ~ 4

Author(s):

Donald D. Lee ◽

Paul D. Sadowski

Keyword(s):

Infected Cell ◽

Dna Synthesis ◽

Base Pair ◽

Bacteriophage T7 ◽

Cell Extracts ◽

In Vitro Incubation ◽

Insight Into

The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome. To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro. Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside [32P]triphosphate resulted in the incorporation of significant radioactivity into the DNA. Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule. Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA. A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.

Download Full-text