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

The Essential Role of the 3' Terminal Template Base in the First Steps of Protein-Primed DNA Replication

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Free PMC article

The Essential Role of the 3' Terminal Template Base in the First Steps of Protein-Primed DNA Replication

Irene Rodríguez et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

Competing Interests: Margarita Salas is a shareholder of X-Pol Biotech. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Formation of the TP-dAMP complex catalysed by either φ29 (A) or Nf (B) DNA polymerase.
Using a single-stranded oligonucleotide with the chemically stable analogue of an abasic site, tetrahydrofuran (THF), at the 3′ end, in the presence of [α32P]dATP, both φ29 and Nf DNA polymerases were able to catalyze the formation of TP-dAMP product. These products were analysed by SDS-PAGE and autoradiography.
Figure 2
Figure 2. (A) The absence of the 3′ base at the template strand makes φ29 DNA polymerase to initiate opposite the third position.
The different initiation products were detected by high resolution SDS-PAGE and analysed by autoradiography. ( B ) Schematic representation of the placement of the 3′ end lacking the terminal base at the active site of φ29 DNA polymerase. On the left side, the φ29 DNA polymerase catalyses the formation of the covalent bond between the initiating dAMP and the hydroxyl group of Ser232 of the TP, using as template the second T at the 3′ end of a wild-type template strand . On the right side, the absence of the terminal base makes the template strand to enter the polymerization domain one position further, leading to the placement of the third 3′ nucleotide at the catalytic site to direct the formation of the initiation complex. (C) The lack of the 3′ terminal base does not alter the initiation position by Nf DNA polymerase. Bands corresponding to TP-dNMP products synthesized by Nf DNA polymerase were detected by high resolution SDS-PAGE.
Figure 3
Figure 3. (A) The absence of the 3′ terminal base at the template oligonucleotide prevents the recovery of the 3′ terminal end by φ29 DNA polymerase.
(B) Size of the products synthesized by φ29 DNA polymerase with the oligonucleotides 3′ T-T-T and 3′ THF-T-T. The length of the product synthesized by φ29 DNA polymerase with the oligonucleotides 3′ T-T-T and 3′ THF-T-T correspond with the intermediate replication products TP-(dNMP)12 and TP-(dNMP)11, respectively, as deduced from the comparison with the products obtained when the natural φ29 TP-DNA is used as template. (C) The lack of the 3′ terminal base at the template strand blocks elongation of the initiation product by Nf DNA polymerase. In all cases, the different elongation products were analysed by high resolution SDS-PAGE. The length at the different products is indicated.

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References

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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.
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