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J Biol Chem. 2015 Nov 6;290(45):27138-45. doi: 10.1074/jbc.M115.682278. Epub 2015 Sep 23.

Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication.

Author information

1
From the Instituto de Biología Molecular "Eladio Viñuela" (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
2
From the Instituto de Biología Molecular "Eladio Viñuela" (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain mdevega@cbm.csic.es.
3
From the Instituto de Biología Molecular "Eladio Viñuela" (Consejo Superior de Investigaciones Científicas), Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain msalas@cbm.csic.es.

Abstract

Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the replication origins, placed at both 5' ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser-232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3' end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser-232 is located indicate that the aromatic residue Phe-230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe-230 in limiting the internalization of the template strand in the polymerization active site is discussed.

KEYWORDS:

DNA polymerase; DNA replication; bacteriophage; nucleic acid; viral polymerase

PMID:
26400085
PMCID:
PMC4646400
DOI:
10.1074/jbc.M115.682278
[Indexed for MEDLINE]
Free PMC Article

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