The essential role of the 3' terminal template base in the first steps of protein-primed DNA replication

PLoS One. 2012;7(10):e48257. doi: 10.1371/journal.pone.0048257. Epub 2012 Oct 24.

Abstract

Bacteriophages ϕ29 and Nf from Bacillus subtilis start replication of their linear genomes at both ends using a protein-primed mechanism by means of which the DNA polymerase initiates replication by adding dAMP to the terminal protein, this insertion being directed by the second and third 3' terminal thymine of the template strand, respectively. In this work, we have obtained evidences about the role of the 3' terminal base during the initiation steps of ϕ29 and Nf genome replication. The results indicate that the absence of the 3' terminal base modifies the initiation position carried out by ϕ29 DNA polymerase in such a way that now the third position of the template, instead of the second one, guides the incorporation of the initiating nucleotide. In the case of Nf, although the lack of the 3' terminal base has no effect on the initiation position, its absence impairs further elongation of the TP-dAMP initiation product. The results show the essential role of the 3' terminal base in guaranteeing the correct positioning of replication origins at the polymerization active site to allow accurate initiation of replication and further elongation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus Phages / genetics
  • DNA Replication / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Replication Origin / genetics
  • Replication Origin / physiology
  • Templates, Genetic

Substances

  • DNA-Directed DNA Polymerase

Grant support

This work was supported by the Spanish Ministry of Science and Innovation (grants BFU 2008-00215, and Consolider-Ingenio CSD 2007-00015 to MS); and Autonomous Community of Madrid (grant P-MAT-0283-0505 to MS). Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.