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.
Highlights
This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: msalas@cbm.uam.es ¤ Current address: X-Pol Biotech, Cantoblanco, Madrid, Spain. These authors contributed to this work. Some organisms such as bacteriophages and eukaryotic viruses contain replication origins formed by inverted terminal repeats (ITR) and a terminal protein (TP) covalently linked at both 59 ends of their linear genomes [1], [2]
The development of an in vitro replication system with purified proteins and TP-DNA from bacteriophage w29 has been instrumental for the elucidation of this so-called protein-primed mechanism of DNA replication [1], [2]
The genome of w29 is a linear dsDNA of 19285 bp, containing a TP of 31 kDa covalently linked to each 59 end [4] that, together with a 6-bp ITR (39-TTTCAT-59) [5], [6] forms part of a minimal replication origin
Summary
Some organisms such as bacteriophages and eukaryotic viruses contain replication origins formed by inverted terminal repeats (ITR) and a terminal protein (TP) covalently linked at both 59 ends of their linear genomes [1], [2]. Once the replication origins are recognized by the heterodimer formed by the DNA polymerase and a free TP molecule [7], [8], the DNA polymerase catalyses the formation of a covalent bond between the initiating dAMP and the hydroxyl group of Ser232 of the TP, a reaction directed by the second T at the 39 end of the template strand [9].
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