Purification and characterization of Caenorhabditis elegans NTH, a homolog of human endonuclease III: Essential role of N-terminal region
ABSTRACT Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast searching of the Wormbase database retrieved a sequence R10E4.5, highly homologous to the human NTH1. However, the R10E4.5-encoded protein did not have NTH activity, and this was considered to be due to lack of the N-terminal region crucial for the activity. Therefore, we purified the protein encoded by the sequence containing both R10E4.5 and the 117-bp region upstream from it, and found that the protein had the NTH activity. The endogenous CeNTH in the extract of C. elegans showed the same DNA glycosylase activity. Therefore, we concluded that the genuine C. elegans NTH gene is not the R10E4.5 but the sequence containing both R10E4.5 and the 117-bp upstream region. NTH-deficient C. elegans showed no difference from the wild-type in lifespan and was not more sensitive to two oxidizing agents, H2O2 and methyl viologen. This suggests that C. elegans has an alternative DNA glycosylase that repairs pyrimidine bases damaged by these agents. Indeed, DNA glycosylase activity that cleaved thymine glycol containing oligonucleotides was detected in the extract of the NTH-deficient C. elegans.
- SourceAvailable from: Nivedita Chatterjee
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- "mU / G > 5 - foU / A >> 8 - oxoG / G ( Morinaga et al . , 2009 ) . The relative impor - tance of the nth - 1 gene in BER in C . elegans is not yet clear . On one hand , the presence of other DNA - glycosylases has been postulated because no genotoxicant - sensitive phenotypes have been detected in nth - 1 deficient mutants ( Meyer et al . , 2000 ; Morinaga et al . , 2009 ; Hunter et al . , 2012 ) . On the other hand , Den - ver et al . ( 2006 ) reported that deletion of the nth - 1 gene results in a mutation - prone phenotype . Although we did not find any influence of PMK - 1 on nth - 1 gene expres - sion , the levels 8 - OHdG were higher ( Fig . 2B ) in AgNP - treated pmk - 1 mutant worms . Fig . 5 . "
ABSTRACT: The large-scale use of silver nanoparticles (AgNPs) has raised concerns over potential impacts on the environment and human health. We previously reported that AgNP exposure causes an increase in reactive oxygen species, DNA damage, and induction of p38 MAPK and PMK-1 in Jurkat T cells and in Caenorhabditis elegans. To elucidate the underlying mechanisms of AgNP toxicity, here we evaluate the effects of AgNPs on oxidative DNA damage-repair (in human and C. elegans DNA glycosylases hOGG1, hNTH1, NTH-1, and 8-oxo-GTPases-hMTH1, NDX-4) and explore the role of p38 MAPK and PMK-1 in this process. Our comparative approach examined viability, gene expression, and enzyme activities in wild type (WT) and p38 MAPK knock-down (KD) Jurkat T cells (in vitro) and in WT and pmk-1 loss-of-function mutant strains of C. elegans (in vivo). The results suggest that p38 MAPK/PMK-1 plays protective role against AgNP-mediated toxicity, reduced viability and greater accumulation of 8OHdG was observed in AgNP-treated KD cells, and in pmk-1 mutant worms compared with their WT counterparts, respectively. Furthermore, dose-dependent alterations in hOGG1, hMTH1, and NDX-4 expression and enzyme activity, and survival in ndx-4 mutant worms occurred following AgNP exposure. Interestingly, the absence or depletion of p38 MAPK/PMK-1 caused impaired and additive effects in AgNP-induced ndx-4(ok1003); pmk-1(RNAi) mutant survival, and hOGG1 and NDX-4 expression and enzyme activity, which may lead to higher accumulation of 8OHdG. Together, the results indicate that p38 MAPK/PMK-1 plays an important protective role in AgNP-induced oxidative DNA damage-repair which is conserved from C. elegans to humans. Environ. Mol. Mutagen., 2013. © 2013 Wiley Periodicals, Inc.Environmental and Molecular Mutagenesis 03/2014; 55(2). DOI:10.1002/em.21844 · 2.55 Impact Factor
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- "One of the main advantages of animal models is that most of them have relatively short life span and that genetic modification is possible. Along with mammalian models, aging research studies and DNA metabolism include unicellular organisms and multicellular eukaryotes such as D. melanogaster, Caenorhabditis elegans, [82–85], or filamentous fungi like Podospora anserina [80, 86]. Investigations in these models have contributed notably to the understanding of the role of DNA repair mechanisms in the aging process in humans, particularly those taking place in mitochondria. "
ABSTRACT: Knowledge about the different mechanisms underlying the aging process has increased exponentially in the last decades. The fact that the basic mechanisms involved in the aging process are believed to be universal allows the use of different model systems, from the simplest eukaryotic cells such as fungi to the most complex organisms such as mice or human. As our knowledge on the aging mechanisms in those model systems increases, our understanding of human aging and the potential interventions that we could approach rise significantly. Among the different mechanisms that have been implicated in the aging process, DNA repair is one of the processes which have been suggested to play an important role. Here, we review the latest investigations supporting the role of these mechanisms in the aging process, stressing how beneficial the use of different model systems is. We discuss how human genetic studies as well as several investigations on mammalian models and simpler eukaryotic organisms have contributed to a better understanding of the involvement of DNA repair mechanisms in aging.Oxidative Medicine and Cellular Longevity 09/2012; 2012(9):282438. DOI:10.1155/2012/282438 · 3.36 Impact Factor
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- "Although C. elegans is known to possess repair homologs to some of the proteins in mammalian DNA repair pathways (11–14), BER has not been well elucidated. Several C. elegans BER repair enzymes have been identified and characterized, including: UDG for uracil removal (15); the nth1 glycosylase for oxidized pyrimidine base removal (16) and two APEs, APN-1 and EXO-3, for AP site incision and processing (17). In addition to these enzymes, C. elegans also is known to express pol θ, a lesion bypass polymerase and back-up BER polymerase in vertebrate systems (18–21). "
ABSTRACT: The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.Nucleic Acids Research 09/2011; 40(2):670-81. DOI:10.1093/nar/gkr727 · 9.11 Impact Factor