Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.

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Abstract
Incubation of extracts of .vphi.29-infected Bacillus subtilis with [.alpha.-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5''-terminal protein of .vphi.29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the .vphi.29 DNA-protein p3 linkage is hydrolyzed released 5''-dAMP. The reaction with [.alpha.-32P]dATP was strongly inhibited by anti-p3 serum and required the presence of .vphi.29 DNA-protein p3 complex; no reaction took place with proteinase K-treated .vphi.29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5''-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 .mu.M [.alpha.-32P]dATP can be elongated by addition of 40 .mu.M dNTP. Treatment with piperidine of the product elongated in the presence of 2'',3''-dideoxycytidine 5''-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.