Formation of the TP-dAMP complex catalysed by either φ29 (A) or Nf (B) DNA polymerase.

<p>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.</p

(A) The absence of the 3′ base at the template strand makes φ29 DNA polymerase to initiate opposite the third position.

<p>The different initiation products were detected by high resolution SDS-PAGE and analysed by autoradiography. <i>(</i><b><i>B</i></b><i>) Schematic representation of the placement of the 3′ end lacking the terminal base at the active site of</i> φ<i>29 DNA polymerase</i>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048257#pone.0048257-Mndez1" target="_blank">[9]</a>. 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. (<b>C</b>) <i>The lack of the 3′ terminal base does not alter the initiation position by Nf DNA polymerase</i>. Bands corresponding to TP-dNMP products synthesized by Nf DNA polymerase were detected by high resolution SDS-PAGE.</p

(A) The absence of the 3′ terminal base at the template oligonucleotide prevents the recovery of the 3′ terminal end by φ29 DNA polymerase.

<p>(<b>B</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. (<b>C</b>) <i>The lack of the 3′ terminal base at the template strand blocks elongation of the initiation product by Nf DNA polymerase</i>. In all cases, the different elongation products were analysed by high resolution SDS-PAGE. The length at the different products is indicated.</p

Oligonucleotides used as template.

<p>Single-stranded oligonucleotides containing the wild-type and modified sequences of the φ29 or Nf DNA right replication origin were obtained from Sigma. These oligonucleotides were used as template for both the initiation assay (TP-dNMP formation) and the truncated elongation assay. THF indicates the presence of tetrahydrofuran, which is a stable analogue of an abasic site.</p

Isolates were grouped into 114 clusters at the 40% similarity level.

<p>Isolates were grouped into 114 clusters at the 40% similarity level.</p

Simpson’s Index of Diversity (1-D) of isolates from different categories (country and host species) at the similarity levels from 25% to 90%.

<p>Simpson’s Index of Diversity (1-D) of isolates from different categories (country and host species) at the similarity levels from 25% to 90%.</p

Minimal spanning tree constructed based on the MLST profiles of the 313 isolates (Bionumerics V6) and coloured according to the cluster numbers: red, cluster 76; purple, cluster 103; light blue, cluster 30; dark green, cluster 42; green, no array data and white, a mixture of rare cluster number.

<p>Minimal spanning tree constructed based on the MLST profiles of the 313 isolates (Bionumerics V6) and coloured according to the cluster numbers: red, cluster 76; purple, cluster 103; light blue, cluster 30; dark green, cluster 42; green, no array data and white, a mixture of rare cluster number.</p