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In vitro Replication of Bacteriophage φ 29 DNA
Kounosuke Watabe, Meng-Fu Shih, Akio Sugino and Junetsu Ito
Proceedings of the National Academy of Sciences of the United States of America
Vol. 79, No. 17, [Part 1: Biological Sciences] (Sep. 1, 1982), pp. 5245-5248
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/12513
Page Count: 4
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We have been studying the mechanisms of linear DNA replication by using Bacillus bacteriophage φ 29 as a model system. To isolate and characterize the proteins required for φ 29 DNA replication, we have developed a cell-free replication system. A cell-free extract prepared from φ 29-infected Bacillus subtilis catalyzes the semiconservative replication of φ 29 DNA, but only if exogenous φ 29 DNA-protein complex is used as the template. This template consists of linear duplex DNA with a 30,000-dalton terminal protein attached covalently to both 5′ ends. Replication starts nonsimultaneously at or near both ends of the template. The extract also catalyzes the specific binding between dATP and the φ 29 terminal protein. Thus, the in vitro system closely mimics the in vivo replication of φ 29 DNA. This system should allow characterization of the φ 29 DNA replication machinery.
Proceedings of the National Academy of Sciences of the United States of America © 1982 National Academy of Sciences