20 research outputs found
Image_2_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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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
Table_2_The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.DOCX
<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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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
Image_1_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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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
Table_1_The Sole DNA Ligase in Entamoeba histolytica Is a High-Fidelity DNA Ligase Involved in DNA Damage Repair.docx
<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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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
Substrate dependent dimerization of monomeric TvTIMs.
<p>(A) Trace of absorbance at 280 nm of TvTIM1 during Sedimentation Velocity experiment at the upper panel followed by the residuals bitmaps. Symbols correspond to experimental data and lines are the results fitted to the Lamm equation using Sedfit [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141747#pone.0141747.ref028" target="_blank">28</a>]. The lower panel shows continuous (c(s)) distribution of wild-type TvTIM1 (black curve) and monomeric mutants I45A (red) and I45G (blue). The left dashed line indicates monomer position whereas the right one indicates dimer. (B) Oligomeric states of I45A mutant in the presence of increasing concentration of PGH. Continuous (c(s)) distribution of I45A mutants in 20 mM Tris-HCl pH 8.0 plus 50 mM NaCl buffer. The distributions of protein without substrate are shown in black lines; the ones with 20 μM of PGH are shown in red lines, with 250 μM of PGH in blue lines, with 600 μM of PGH in pink lines, with 1000 μM of PGH in green lines and with 2000 μM of PGH in dark blue lines.</p
Unfolding profiles of TvTIM1 and I45G mutant monitored by Intrinsic (black squares) and ANS (red circles) fluorescence
<p>. The conditions are as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141747#pone.0141747.g004" target="_blank">Fig 4</a>. The protein concentration was 50 μg ml<sup>-1</sup>. Data are normalized for ease of comparison.</p
Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from <i>Trichomonas vaginalis</i>
<div><p>The dimeric nature of triosephosphate isomerases (TIMs) is maintained by an extensive surface area interface of more than 1600 Å<sup>2</sup>. TIMs from <i>Trichomonas vaginalis</i> (TvTIM) are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A) is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.</p></div
Ball and socket interaction between monomers at TvTIM1.
<p><b>(A)</b> Crystal structure of TvTIM1 showing the ball and socket interplay. Hydrophobic TvTIM1 residues (ball-stick representation) form a socket that interacts with residue Ile45 of the neighboring subunit (space-filling representation). The methyl group of I45 is colored in black. <b>(B)</b> Sequence logo showing the structural alignment of the ball-socket amino acids at the ball and socket interplay Residue I45 or V45 functions as the ball and a hydrophobic cavity formed by α-helices 2 and 3 that assemble as the socket.</p
<i>In vivo</i> characterization of TIMs complemented strains on glycerol minimal media in the presence of IPTG.
<p><b>(A)</b> Complementation of point mutants into an <i>E</i>. <i>coli</i> DE3 ΔTIM strain. Transformed <i>E</i>. <i>coli</i> were grown in plates with glycerol as a carbon source in M63 minimal media and 0.1mM of IPTG. <i>E</i>. <i>coli</i> transformed with plasmids containing wild-type TvTIM1 are able to complement. Mutants I45A, I45V, I45F and I45L complement with similar efficiency as wild type, whereas mutants I45Y contained less colonies, and no colonies appear after 48 hrs for I45G and I445W. <b>(B)</b> Growth kinetics of <i>E</i>. <i>coli</i> complemented strains in liquid minimal media. Growth rates of cultures grown in minimal medium. Bacteria transformed with plasmids containing wild-type TvTIM1 and mutants I45L, I45V, complement with similar efficiency, whereas, mutants I45A, I45F, I45Y and I45G present slower growth rates and no growth was observed with the tryptophan mutant and the empty vector.</p
AUC data analysis for I45A mutant in 20 mM Tris-HCl.
<p>pH 8.0 and 50 mM NaCl with different concentrations of PGH</p><p>AUC data analysis for I45A mutant in 20 mM Tris-HCl.</p