Overproduction and Purification of the Gene 2 Product Involved in the Initiation of Phage Φ29 Replication
The Bacillus subtilis phage Φ29 has a linear, double-stranded DNA with the protein product of gene 3, p3, covalently linked to the two 5’ ends (1). Φ29 replication starts at either DNA end by a novel mechanism by which the terminal protein p3 primes replication by reaction with dATP and formation of a protein p3-dAMP covalent complex that provides the 3’0H group needed for elongation by the DNA polymerase. The initiation reaction, i.e. the formation of the protein p3-dAMP covalent complex, requires Φ29 DNA-protein p3 as a template, free protein p3 and the gene 2 product (2, 3). We present here the cloning of gene 2 and the overproduction and partial purification of protein p2. A DNA polymerase activity has been found associated with protein p2.
KeywordsAmmonium Sulfate Partial Purification Initiation Complex Initiation Reaction Plasmid pBR322
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