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.
Highlights
Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, must cope with reactive oxygen (ROS) and nitric oxide (NOS) species derived from human immune cells, during colonic tissue invasion and metronidazole drug treatment (Vicente et al, 2009; Wilson et al, 2012)
We found that rEhDNAligI was able to discriminate almost all the noncanonical Watson-Crick base pairs either at the 3′-OH or 5′PO4 of the nicked DNA. rEhDNAligI only efficiently ligated the T:G mismatch at the 5′- PO4 strand (Figure 1A, lane 20)
DNA ligation kinetics for a G:T mismatch in comparison to ligation employing G:C and T:A canonical pairs indicated that the mismatch and the canonical pairs are ligated with similar catalytic efficiencies that differ by no more than twofold in their Km
Summary
The protozoan parasite responsible for human amoebiasis, must cope with reactive oxygen (ROS) and nitric oxide (NOS) species derived from human immune cells, during colonic tissue invasion and metronidazole drug treatment (Vicente et al, 2009; Wilson et al, 2012). 8-oxoG is recognized and processed by the nucleotide excision repair (NER) pathway, suggesting a cross talk between NER and BER to repair non-bulky oxidative DNA lesions (Menoni et al, 2012; Parlanti et al, 2012). In eukaryotic cells the interactions between DNA ligase I with PCNA and DNA polymerase β are critical for Okazaki fragment maturation and DNA repair, respectively (Dianov et al, 1998). DNA ligase I is recruited to UV-damage sites only in proliferating cells and it is implicated in BER and NER pathways (Moser et al, 2007). DNA ligase III is involved in NER following UV-damage in quiescent cells, singlestrand, and double stranded break repair; whereas DNA ligase IV is involved in DNA double-strand breaks repaired by the NHEJ pathway (Tomkinson et al, 2006)
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