PROTEIN NUCLEIC ACID INTERACTIONS IN BACTERIOPHAGE-PHI-29 DNA-REPLICATION

phi 29 DNA replication starts at both DNA ends by a protein priming me chanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongatio n has been proposed to occur by a sliding-back mechanism that is neces sary for maintaining the sequences at the phi 29 DNA ends. Structure-f unction studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly re lated DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the stra nd-displacement capacity, whereas the C-terminal domain contains the s ynthetic activities (protein-primed initiation and DNA polymerization) . Viral protein p6 stimulates the initiation of phi 29 DNA replication . The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an a-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. Th e phi 29 protein p5 is the single-stranded DNA binding (SSB) protein i nvolved in phi 29 DNA replication, by binding to the displaced single- stranded DNA (ssDNA) in the replication intermediates. In addition, pr otein p5 is able to unwind duplex DNA. The properties of the phi 29 SS B-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possi ble to amplify the 19 285-bp phi 29 DNA molecule by a factor of 4000 a fter 1 h of incubation at 30 degrees C. The infectivity of the in vitr o amplified DNA was identical to that of phi 29 DNA obtained from viri ons.