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Closed Access item Protein—nucleic acid interactions in bacteriophage φ29 DNA replication

Authors:Salas, Margarita
Hermoso, José A.
Keywords:DNA polymerase, Nucleoprotein complex, Phage φ29, Protein-primed replication, SSB protein, Terminal protein
Issue Date:1995
Publisher:John Wiley & Sons
Citation:FEMS Microbiology Reviews 17(1-2): 73-82) 1995
Abstract:φ29 DNA replication starts at both DNA ends by a protein priming mechanism. 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 elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3′–5′ exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions.
Publisher version (URL):http://dx.doi.org/10.1111/j.1574-6976.1995.tb00189.x
URI:http://hdl.handle.net/10261/40600
ISSN:1574-6976
10.1111/j.1574-6976.1995.tb00189.x
Appears in Collections:(CBM) Artículos

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