ΔTPR2 bypasses 8oxoG, but not an abasic site.

<p>Lesion bypass of EhDNApolB2 (lanes 1 to 14) and ΔTPR2 (lanes 15 to 28). The time course primer extension is described as in material and methods using equal amounts of DNA polymerases and 100 µM dNTPs. After incubation times of 2.5, 5, 10 and 20 minutes the primer extension reactions were stopped and run onto a 15% denaturing polyacrylamide gel.</p

EhDNApolB2 efficiently bypasses 8-oxo guanosine and abasic site lesions.

<p>Denaturing polyacrylamide gel electrophoresis showing translesion bypass of EhDNApolB2 in comparison to undamaged template. Primer extension by EhDNApolB2 using a canonical and damaged substrate. The first nucleotide (canonical or damaged) that serves a template is designated by an <b>X</b>. For the 8-oxoguanosine and abasic site the lesion is located immediately after a primer of 29 nt and for thymine glycol and UV adducts is located immediately after a primer of 16 nt. The label 25, 30 or 17 nt indicate the length of the primer is only one nucleotide opposite the lesion is incorporated. Each reaction was incubated with a 20 nM of EhDNApolB2 and 1 nM of several substrates. Aliquots were taken at 0, 2.5, 5, 10 and 20 minutes. Time course of different substrates were loaded in a 15% denaturing gel. Thymine (lanes 1–5); 8-oxo guanosine (lanes 6–10); abasic site (lanes 11–15); 5 S-6R thymine glycol (lanes 16–20); 5R-6S thymine glycol (lanes 21–24); cis-syn cyclobutane pyrimidine dimer (lanes 25–29); 6-4 photo product (lanes 30–33); The upper arrow depicts the length of the final product substrate and the bottom arrow indicates the used primer.</p

Processivity of EhDNApolB2 in comparison to φ29 DNA polymerase, and its dependence on TPR2.

<p>The processivity of wild-type EhDNApolB2 was measured in comparison to φ29 DNA polymerase and ΔTPR2. Reactions were carried out with 20 nM of the indicated polymerase and 1 nM of γ–P³²17mer primer annealed to circular M13mp18 ssDNA. Aliquots were taken at 10, 20 and 40 min and loaded onto a 6% denaturing polyacrylamide gel. The arrows in the right correspond to the full-length M13 DNA amplification and abortive products.</p

TPR2 is required for efficient strand-displacement.

<p>Strand displacement was assessed using a set of 3 oligonucleotides with gaps of 1, 3 and 6 nt respectively. After incubation at the indicated times the reaction mixtures were run on a 18% denaturing polyacrylamide gel. Reactions were carried out in 20 µl as described in material in methods (<b>A</b>) <b>Strand-displacement activity of EhDNApolB2</b>. Primer extension (lanes 2 to 5), primer extension with 1 nt gap (lanes 6 to 9), primer extension with 3 nt gap (lanes 10 to 13), primer extension with 6 nt gap (14 to 17). (<b>B</b>) <b>Strand-displacement activity of</b> Δ<b>TPR2</b> Primer extension (lanes 2 to 5), primer extension with 1 nt gap (lanes 6 to 9), primer extension with 3 nt gap (lanes 10 to 13), primer extension with 6 nt gap (14 to 17).</p

Modular organization of family B2 DNA polymerases in <i>E. histolytica</i> and structural model of EhDNApolB2.

<p>(<b>A</b>) <i>E. histolytica</i> contains four family B2 DNA polymerases in its genome. These DNA polymerase present a C-terminal region with conserved exonuclease and polymerase motifs characteristic of a family B2 DNA polymerases (green, blue and red boxes). The N-terminal region, indicated by a thin line, presents no similitude to other proteins and is composed of 180 to 500 amino acids. The shortest DNA polymerase is present at loci EHI_018010 and is dubbed EhDNApolB2 throughout this work (<b>B</b>) Homology structural model of EhDNApolB2. The 3′–5′ exonuclease domain is shown in green and the 5′–3′ polymerization domain is shown in blue. The extended TPR2 motif is shown in red encircling double stranded DNA (yellow colored).</p

TPR2 is responsible of lesion bypass extension opposite an abasic site.

<p>Lesion bypass of wtEhDNApolB2 (lanes 1 to 5 and 11 to 15) and ΔTPR2 (lanes 6 to 10 and 16 to 20) extending from a primer containing a 3′OH purine or a pyrimidine opposite an abasic site. A primer containing a 3′OH dAMP (lanes 1 to 10) or dCMP (11 to 20) opposite an abasic site was subject to a time course primer extension reaction from 2.5 to 20 minutes using equal amounts of DNA polymerases and 100 µM dNTPs. The reaction products were run onto a 15% denaturing polyacrylamide gel.</p

Fidelity of translesion DNA synthesis of EhDNApolB2.

<p>Translesion bypass fidelity of EhDNApolB 20 nM of exonuclease deficient EhDNApolB were incubated with 1 nM of a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. The dNTPs were present at a concentration of 15 µM. Samples were taken at 2.5 minutes, stopped with 50 mM EDTA and 90% formamide and run onto a 18% denaturing polyacrylamide gel electrophoresis for their analysis by phosphorimagery. (A) Control thymine (lanes 1 to 5), 8-oxo guanosine (lanes 6 to 10), and abasic site (lanes 11 to 15). (B) 5 S-6R and 5R-6S thymine glycol (lanes 1 to 5 and 6 to 10 respectively). The upper arrow depicts the length of the final product substrate and the bottom arrow indicates the used primer.</p