Protein—nucleic acid interactions in bacteriophage φ 29 DNA replication

Abstract

~b29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the tenrninai protein-dAMP initiation complex is directed by the second nueleotide from the 3' end of the template. The transiti~m from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism tthat is necessary for maintaining the sequences at the ~b29 DNA ends. Structure-function studies have been carried out in the ~k29 DNA polymerase, By site-directed mutagenesis of amino acids conserved among distantly related DNA polymer~s~es we have shown that the N-terminal domain of ~b29 DNA polymerase contains the 3'-5' exonuclease activity ~atd the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiati00n and DNA polymerization). Viral protein p6 stimulates the initiation of ~b29 DNA replication. The structure of the p~rotein p6-DNA complex has been determined, as well as the main signals at the 029 DNA ends recognized by protein :~. The DNA binding domain of protein p6 has been studied. The results indicate that an o~-helical structure located fin the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The ~b29 protein p5 iis the single-stranded DNA binding (SSB) protein involved in ~b29 DNA replication, by binding to the displaced single-slrzanded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties ,of the ~b29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p~6 and the SSB protein, it was possible to amplify the 19 285-bp 029 DNA molecule by a factor of 4000 after 1 h of ineul~a~ion at 30°C. The infectivity of the in vitro amplified DNA was identical to that of q~29 DNA obtained from virions.