In vitro replication of bacteriophage phi 29 DNA.

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Abstract
The mechanisms of linear DNA replication were studied by using Bacillus bacteriophage .vphi.29 as a model system. To isolate and characterize the proteins required from .vphi.29 DNA replication, a cell-free replication system was developed. A cell-free extract prepared from .vphi.29-infected Bacillus subtilis catalyzes the semiconservative replication of .vphi.29 DNA, but only if exogenous .vphi.29 DNA.sbd.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 .vphi.29 terminal protein. Thus, the in vitro system closely mimics the in vivo replication of .vphi.29 DNA. This system should allow characterization of the .vphi.29 DNA replication machinery.