Bacteriophage T7 Deoxyribonucleic Acid Replication in Vitro

• Published in: The Journal of biological chemistry 1974-05, Vol.249 (9), p.2974-2980
• Main Authors: Hinkle, David C., Richardson, Charles C.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)42726-9

A cell-free system for the study of bacteriophage T7 DNA replication has been developed. DNA synthesis in vitro requires the four deoxynucleoside triphosphates and an exogenous T7 DNA template. The rate of synthesis is increased 5- to 6-fold by the four ribonucleoside triphosphates. The products of the phage gene 4 and gene 5 (DNA polymerase), but not those of genes 2, 3, and 6, are required for DNA synthesis in vitro. The kinetics of synthesis is linear for 20 to 30 min, and 30 min after the start of the reaction the amount of DNA synthesized is 2 to 3 times the amount of template DNA added to the reaction. The product is not covalently attached to the template. Analysis by zone sedimentation through alkaline sucrose gradients indicates that the products of synthesis are short (11 S) DNA chains, suggesting that the sealing of “Okazaki pieces” into DNA of high molecular weight is inefficient in this in vitro reaction. Examination of the product in the electron microscope reveals many large, forked molecules, indicating that in some cases large portions of the T7 genome are replicated in vitro.