The sole DNA ligase in entamoeba histolytica is a high-fidelity DNA ligase involved in DNA damage repair

"The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolyticaencodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H2O2 treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H2O2 treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.

A transposon-derived DNA polymerase from Entamoeba histolytica displays intrinsic strand displacement, processivity and lesion bypass.

Entamoeba histolytica encodes four family B2 DNA polymerases that vary in amino acid length from 813 to 1279. These DNA polymerases contain a N-terminal domain with no homology to other proteins and a C-terminal domain with high amino acid identity to archetypical family B2 DNA polymerases. A phylogenetic analysis indicates that these family B2 DNA polymerases are grouped with DNA polymerases from transposable elements dubbed Polintons or Mavericks. In this work, we report the cloning and biochemical characterization of the smallest family B2 DNA polymerase from E. histolytica. To facilitate its characterization we subcloned its 660 amino acids C-terminal region that comprises the complete exonuclease and DNA polymerization domains, dubbed throughout this work as EhDNApolB2. We found that EhDNApolB2 displays remarkable strand displacement, processivity and efficiently bypasses the DNA lesions: 8-oxo guanosine and abasic site.Family B2 DNA polymerases from T. vaginalis, G. lambia and E. histolytica contain a Terminal Region Protein 2 (TPR2) motif twice the length of the TPR2 from φ29 DNA polymerase. Deletion studies demonstrate that as in φ29 DNA polymerase, the TPR2 motif of EhDNApolB2 is solely responsible of strand displacement and processivity. Interestingly the TPR2 of EhDNApolB2 is also responsible for efficient abasic site bypass. These data suggests that the 21 extra amino acids of the TPR2 motif may shape the active site of EhDNApolB2 to efficiently incorporate and extended opposite an abasic site. Herein we demonstrate that an open reading frame derived from Politons-Mavericks in parasitic protozoa encode a functional enzyme and our findings support the notion that the introduction of novel motifs in DNA polymerases can confer specialized properties to a conserved scaffold

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

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

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

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

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

Image_3_The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.JPEG

<p>The protozoan parasite Entamoeba histolytica is exposed to reactive oxygen and nitric oxide species that have the potential to damage its genome. E. histolytica harbors enzymes involved in DNA repair pathways like Base and Nucleotide Excision Repair. The majority of DNA repairs pathways converge in their final step in which a DNA ligase seals the DNA nicks. In contrast to other eukaryotes, the genome of E. histolytica encodes only one DNA ligase (EhDNAligI), suggesting that this ligase is involved in both DNA replication and DNA repair. Therefore, the aim of this work was to characterize EhDNAligI, its ligation fidelity and its ability to ligate opposite DNA mismatches and oxidative DNA lesions, and to study its expression changes and localization during and after recovery from UV and H₂O₂ treatment. We found that EhDNAligI is a high-fidelity DNA ligase on canonical substrates and is able to discriminate erroneous base-pairing opposite DNA lesions. EhDNAligI expression decreases after DNA damage induced by UV and H₂O₂ treatments, but it was upregulated during recovery time. Upon oxidative DNA damage, EhDNAligI relocates into the nucleus where it co-localizes with EhPCNA and the 8-oxoG adduct. The appearance and disappearance of 8-oxoG during and after both treatments suggest that DNA damaged was efficiently repaired because the mainly NER and BER components are expressed in this parasite and some of them were modulated after DNA insults. All these data disclose the relevance of EhDNAligI as a specialized and unique ligase in E. histolytica that may be involved in DNA repair of the 8-oxoG lesions.</p