The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair

Abstract

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.

Epithelial Cells Expressing EhADH, An Entamoeba histolytica Adhesin, Exhibit Increased Tight Junction Proteins

“…New Zealand male rabbits were immunized three times (each 2 weeks) with 300 μg of this peptide diluted in TiterMax® Gold Adjuvant liquid (Sigma). Other primary antibodies used were: mouse against EhADH (mAbAdh) (Arroyo and Orozco, 1987 ), α-actin (kindly donated by Dr. José Manuel Hernández from Department of Cellular Biology, CINVESTAV, Mexico), α-claudin-1 (Invitrogen), α-occludin (Invitrogen) and α-caveolin-1 (Santa Cruz Biotechnology); rabbit α-ZO-2 (Invitrogen), α-ZO-1 (Invitrogen), α-occludin (Invitrogen), α-α/β tubulin (Cell Signaling), and α-PCNA (Azuara-Liceaga et al, 2018 ); and goat α-clathrin (Santa Cruz Biotechnology) and α-GAPDH (Santa Cruz Biotechnology). For some experiments, mouse IgM isotype control (Thermo Fisher) was used.…”

Episomal and chromosomal DNA replication and recombination in Entamoeba histolytica

“…In contrast to vertebrates, E. histolytica encodes a single DNA ligase (EhDNAligI) which is similar to eukaryotic DNA ligase I ( Azuara-Liceaga et al, 2018 ). It is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I ( Cardona-Felix et al, 2010 ).…”

“…It was a high-fidelity DNA ligase and could discriminate 11 of the 12 possible mismatches on either side of the nick on the two DNA strands. The only mismatch efficiently ligated was the T:G mismatch at the 5′-phosphate strand ( Azauara-Liceaga et al, 2018 ). This wobble base-pair mismatch is seen in ligases of Escherichia coli and Thermus thermophilus as well ( Chauleau and Shuman, 2016 ).…”

Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review

“…Two types of ligases have been found to catalyze nucleic acid ligations: DNA ligase and RNA ligase. DNA ligase forms a phosphodiester bond by connecting the adjacent 5’-terminal phosphate group with the 3’-terminal hydroxyl group on the catalytic chain so that the single-stranded nick can be repaired, which is known as “the needle and thread of genes” [ 6 ]. Ligase can be divided into room-temperature and thermophilic DNA ligases based on the thermal stability of DNA ligase.…”