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Miral Dizdaroglu - One of the best experts on this subject based on the ideXlab platform.
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identification and quantification of DNA Repair Protein apurinic apyrimidinic endonuclease 1 ape1 in human cells by liquid chromatography isotope dilution tandem mass spectrometry
PLOS ONE, 2013Co-Authors: Güldal Kirkali, Pawel Jaruga, David M Wilson, Prasad T. Reddy, Bryant C. Nelson, Alessandro Tona, Mengxia Li, Miral DizdarogluAbstract:Unless Repaired, DNA damage can drive mutagenesis or cell death. DNA Repair Proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA Repair Protein expression and genotype is of fundamental importance. Among DNA Repair Proteins involved in base excision Repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely 15N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and 15N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant Protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this Protein in disease development and treatment responses.
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abstract 618 identification and quantification of human DNA Repair Protein neil1 by liquid chromatography isotope dilution tandem mass spectrometry as a potential cancer biomarker
Cancer Research, 2013Co-Authors: Miral DizdarogluAbstract:Accumulated evidence points to DNA Repair capacity as an important therapeutic factor in cancer in predicting patient response to DNA-damaging agents such as chemotherapeutic drugs and ionizing radiation. Recent findings suggest that some types of malignant tumors possess increased DNA Repair capacity that may affect the therapy and outcome of cancer. Thus, the knowledge of the over-expression or under-expression levels of DNA Repair Proteins in tumors and disease-free tissues will help develop and guide treatment strategies that will likely lead to the best treatment results for patients. In this context, DNA Repair Proteins are becoming predictive, prognostic and therapeutic factors in cancer, and also promising drug targets for cancer treatment as DNA Repair inhibitors are being developed to increase the efficacy of cancer therapy. We developed assays for the mass spectrometric measurement in tissues of the human DNA Repair Protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision Repair pathways of oxidatively induced DNA damage. There is strong evidence for a critical role of NEIL1 in maintaining the genetic stability and in prevention of diseases such as cancer and metabolic syndrome. We applied liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), using the fully 15N-labeled analogue of hNEIL1 (15N-hNEIL1) as an internal standard, which we produced, purified and characterized. Both hNEIL1 and 15N-hNEIL1 were hydrolyzed with trypsin. Eighteen tryptic peptides of each Protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1, and provided a statistically significant Protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides of both Proteins were recorded and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and 15N-hNEIL1 on the basis of product ions. We also showed the detection of these Proteins following their separation by gel electrophoresis and in-gel trypsin digestion. Our results suggest that the assays developed would be highly suitable for the positive identification and accurate quantification of hNEIL1 in tissues in vivo as a potential cancer biomarker. . Citation Format: Miral Dizdaroglu. Identification and quantification of human DNA Repair Protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 618. doi:10.1158/1538-7445.AM2013-618
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identification and quantification of human DNA Repair Protein neil1 by liquid chromatography isotope dilution tandem mass spectrometry
Journal of Proteome Research, 2013Co-Authors: Prasad T. Reddy, Pawel Jaruga, Güldal Kirkali, Gamze Tuna, Bryant C. Nelson, Miral DizdarogluAbstract:Accumulated evidence points to DNA Repair capacity as an important factor in cancer and other diseases. DNA Repair Proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA Repair Proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA Repair Protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision Repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), in combination with a purified and fully characterized recombinant (15)N-labeled analogue of hNEIL1 ((15)N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and (15)N-hNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each Protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant Protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both Proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and (15)N-hNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the Proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC-MS/MS analysis. These results suggest that the developed assays would be highly suitable for the in vivo positive identification and accurate quantification of hNEIL1 in tissues.
Alfonso Bellacosa - One of the best experts on this subject based on the ideXlab platform.
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interaction with the DNA Repair Protein thymine DNA glycosylase regulates histone acetylation by p300
Biochemistry, 2016Co-Authors: Ryan A Henry, Rossella Tricarico, Pietro Mancuso, Marc Tini, Philip A. Cole, Alfonso Bellacosa, Andrew J AndrewsAbstract:How Protein–Protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other Proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA Repair/demethylation.
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biphasic kinetics of the human DNA Repair Protein med1 mbd4 a mismatch specific DNA n glycosylase
Journal of Biological Chemistry, 2000Co-Authors: Fiorella Petronzelli, Antonio Riccio, Steven H Seeholzer, Jay Stoerker, George D Markham, Maurizio Genuardi, Anthony T. Yeung, Yoshihiro Matsumoto, Alfonso BellacosaAbstract:Abstract The human Protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch Repair Protein MLH1 as a bait, and shown to have homology to bacterial base excision Repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.
Giovanni Neri - One of the best experts on this subject based on the ideXlab platform.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.
Jing Gung Chung - One of the best experts on this subject based on the ideXlab platform.
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alpha phellandrene induced DNA damage and affect DNA Repair Protein expression in wehi 3 murine leukemia cells in vitro
Environmental Toxicology, 2015Co-Authors: Chih Chung Wu, Yi Shih, Shuwen Weng, Yiping Huang, Jing Gung ChungAbstract:Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA Repair associated Protein expression. Herein, we investigated the effects of α-PA on DNA damage and Repair associated Protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and Repair associated Protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 Protein expression but inhibited the Protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1322–1330, 2015.
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alpha phellandrene induced DNA damage and affect DNA Repair Protein expression in wehi 3 murine leukemia cells in vitro
Environmental Toxicology, 2015Co-Authors: Jen Jyh Lin, Yi Shih, Shuwen Weng, Yiping Huang, Jaung-geng Lin, Shu Chun Hsu, Jing Gung ChungAbstract:Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA Repair associated Protein expression. Herein, we investigated the effects of α-PA on DNA damage and Repair associated Protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and Repair associated Protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 Protein expression but inhibited the Protein of p53, MGMT, DNA-PK, and BRCA-1.
Yoshihiro Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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The interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites
Journal of Biological Chemistry, 2012Co-Authors: Julie Della-maria, Muralidhar L. Hegde, Daniel R. Mcneill, Miaw Sheue Tsai, Tom Ellenberger, Sankar Mitra, Yoshihiro Matsumoto, David M Wilson, Alan E. TomkinsonAbstract:XRCC1 plays a key role in the Repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA Repair Proteins and is a subunit of distinct DNA Repair Protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single Protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner Proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted Repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage Repair.
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biphasic kinetics of the human DNA Repair Protein med1 mbd4 a mismatch specific DNA n glycosylase
Journal of Biological Chemistry, 2000Co-Authors: Fiorella Petronzelli, Antonio Riccio, Steven H Seeholzer, Jay Stoerker, George D Markham, Maurizio Genuardi, Anthony T. Yeung, Yoshihiro Matsumoto, Alfonso BellacosaAbstract:Abstract The human Protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch Repair Protein MLH1 as a bait, and shown to have homology to bacterial base excision Repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.
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med1 a novel human methyl cpg binding endonuclease interacts with DNA mismatch Repair Protein mlh1
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Alfonso Bellacosa, Lucia Cicchillitti, Antonio Riccio, Erica A. Golemis, Maurizio Genuardi, Anthony T. Yeung, Filippo Schepis, Yoshihiro Matsumoto, Giovanni NeriAbstract:The DNA mismatch Repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR Protein MLH1 as bait, we cloned MED1. The MED1 Protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA Repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA Repair Protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA Repair.