The Experts below are selected from a list of 47004 Experts worldwide ranked by ideXlab platform
Sankar Mitra - One of the best experts on this subject based on the ideXlab platform.
-
Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress.
DNA repair, 2018Co-Authors: Shiladitya Sengupta, Muralidhar L Hegde, Kishor K Bhakat, Pavana M Hegde, Chunying Yang, Joy Mitra, Arvind Pandey, Arijit Dutta, Abdul Tayyeb Datarwala, Sankar MitraAbstract:Abstract Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1’s disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296–298 with neutral Ala loses nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites does not affect NEIL1’s nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing the acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.
-
Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A
Nucleic acids research, 2016Co-Authors: Chunying Yang, Muralidhar L Hegde, Pavana M Hegde, Shiladitya Sengupta, Joy Mitra, Shuai Jiang, Brooke Holey, Altaf H. Sarker, Miaw-sheue Tsai, Sankar MitraAbstract:Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.
-
prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 dna glycosylase together with replication proteins
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Muralidhar L Hegde, Alan E Tomkinson, Istvan Boldogh, Tapas K Hazra, Pavana M Hegde, Larry J Bellot, Santi M Mandal, Guo Min Li, Sankar MitraAbstract:Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.
-
The Disordered C-Terminal Domain of Human DNA Glycosylase NEIL1 Contributes to Its Stability via Intramolecular Interactions
Journal of molecular biology, 2013Co-Authors: Muralidhar L Hegde, Pavana M Hegde, Susan E. Tsutakawa, Luis Marcelo F. Holthauzen, Numan Oezguen, Vincent J. Hilser, John A. Tainer, Sankar MitraAbstract:Abstract NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~ 100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355–360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions.
-
human dna glycosylase NEIL1 s interactions with downstream repair proteins is critical for efficient repair of oxidized dna base damage and enhanced cell survival
Biomolecules, 2012Co-Authors: Muralidhar L Hegde, Istvan Boldogh, Pavana M Hegde, Dutta Arijit, Sankar MitraAbstract:NEIL1 is unique among the oxidatively damaged base repair-initiating DNA glycosylases in the human genome due to its S phase-specific activation and ability to excise substrate base lesions from single-stranded DNA. We recently characterized NEIL1’s specific binding to downstream canonical repair and non-canonical accessory proteins, all of which involve NEIL1’s disordered C-terminal segment as the common interaction domain (CID). This domain is dispensable for NEIL1’s base excision and abasic (AP) lyase activities, but is required for its interactions with other repair proteins. Here, we show that truncated NEIL1 lacking the CID is markedly deficient in initiating in vitro repair of 5-hydroxyuracil (an oxidative deamination product of C) in a plasmid substrate compared to the wild-type NEIL1, thus suggesting a critical role of CID in the coordination of overall repair. Furthermore, while NEIL1 downregulation significantly sensitized human embryonic kidney (HEK) 293 cells to reactive oxygen species (ROS), ectopic wild-type NEIL1, but not the truncated mutant, restored resistance to ROS. These results demonstrate that cell survival and NEIL1-dependent repair of oxidative DNA base damage require interactions among repair proteins, which could be explored as a cancer therapeutic target in order to increase the efficiency of chemo/radiation treatment.
Miral Dizdaroglu - One of the best experts on this subject based on the ideXlab platform.
-
Enhanced sensitivity of NEIL1-/- mice to chronic UVB exposure.
DNA repair, 2016Co-Authors: Marcus J. Calkins, Amanda K Mccullough, Pawel Jaruga, Miral Dizdaroglu, Güldal Kirkali, Vladimir Vartanian, Nichole Owen, R. Stephen LloydAbstract:Abstract Oxidative stress and reactive oxygen species (ROS)-induced DNA base damage are thought to be central mediators of UV-induced carcinogenesis and skin aging. However, increased steady-state levels of ROS-induced DNA base damage have not been reported after chronic UV exposure. Accumulation of ROS-induced DNA base damage is governed by rates of lesion formation and repair. Repair is generally performed by Base Excision Repair (BER), which is initiated by DNA glycosylases, such as 8-oxoguanine glycosylase and Nei-Endonuclease VIII-Like 1 (NEIL1). In the current study, UV light (UVB) was used to elicit protracted low-level ROS challenge in wild-type (WT) and NEIL1 −/− mouse skin. Relative to WT controls, NEIL1 −/− mice showed an increased sensitivity to tissue destruction from the chronic UVB exposure, and corresponding enhanced chronic inflammatory responses as measured by cytokine message levels and profiling, as well as neutrophil infiltration. Additionally, levels of several ROS-induced DNA lesions were measured including 4,6-diamino-5-formamidopyrimidine (FapyGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyAde), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxyuracil (5,6-diOH-Ura) and thymine glycol (ThyGly). In WT mice, chronic UVB exposure led to increased steady-state levels of FapyGua, FapyAde, and ThyGly with no significant increases in 8-OH-Gua or 5,6-diOH-Ura. Interestingly, the lesions that accumulated were all substrates of NEIL1. Collectively, these data suggest that NEIL1-initiated repair of a subset of ROS-induced DNA base lesions may be insufficient to prevent the initiation of inflammatory pathways during chronic UV exposure in mouse skin.
-
abstract 2421 small molecule inhibitors of dna glycosylases as potential drugs in cancer therapy
Cancer Research, 2014Co-Authors: Miral Dizdaroglu, Pawel JarugaAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA glycosylases are required to initiate the DNA base excision repair pathway of lesions that are formed by DNA damage. Recently, several DNA glycosylases including NEIL1, OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies for cancer treatment. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. In this work, we designed experimental strategies to discover small molecule inhibitors of NEIL1, with subsequent analyses of the specificity of those molecules for other DNA glycosylases. Using NEIL1 as the proof-of-principle DNA glycosylase, we developed a fluorescence-based assay that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of purine-derived lesions. We also applied gas chromatography/isotope-dilution tandem mass spectrometry for the measurement of the effect of preferred small molecule inhibitors on activities of the DNA glycosylases NEIL1, OGG1 and NTH1. The release of modified bases, which are known substrates of these enzymes, was measured in the presence and absence of the inhibitors. These data revealed that several of the purine analogs were general glycosylase inhibitors with one compound being the most effective inhibitor for all three enzymes. However, there were significance differences in inhibition of enzymatic activities among these three DNA glycosylases. Overall, this work forms the foundation for the future discovery of DNA repair inhibitors as drugs in cancer therapy for the entire family of DNA glycosylases. Citation Format: Miral Dizdaroglu, Pawel Jaruga. Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2014-2421
-
abstract 2421 small molecule inhibitors of dna glycosylases as potential drugs in cancer therapy
Cancer Research, 2014Co-Authors: Miral Dizdaroglu, Pawel JarugaAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA glycosylases are required to initiate the DNA base excision repair pathway of lesions that are formed by DNA damage. Recently, several DNA glycosylases including NEIL1, OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies for cancer treatment. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. In this work, we designed experimental strategies to discover small molecule inhibitors of NEIL1, with subsequent analyses of the specificity of those molecules for other DNA glycosylases. Using NEIL1 as the proof-of-principle DNA glycosylase, we developed a fluorescence-based assay that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of purine-derived lesions. We also applied gas chromatography/isotope-dilution tandem mass spectrometry for the measurement of the effect of preferred small molecule inhibitors on activities of the DNA glycosylases NEIL1, OGG1 and NTH1. The release of modified bases, which are known substrates of these enzymes, was measured in the presence and absence of the inhibitors. These data revealed that several of the purine analogs were general glycosylase inhibitors with one compound being the most effective inhibitor for all three enzymes. However, there were significance differences in inhibition of enzymatic activities among these three DNA glycosylases. Overall, this work forms the foundation for the future discovery of DNA repair inhibitors as drugs in cancer therapy for the entire family of DNA glycosylases. Citation Format: Miral Dizdaroglu, Pawel Jaruga. Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2014-2421
-
Inhibition of DNA glycosylases via small molecule purine analogs.
PLOS ONE, 2013Co-Authors: Aaron C. Jacobs, Marcus J. Calkins, Amanda K Mccullough, Dorjbal Dorjsuren, Anton Simeonov, Ajit Jadhav, David G Maloney, Pawel Jaruga, Miral Dizdaroglu, R. Stephen LloydAbstract:Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.
-
Abstract 618: Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry as a potential cancer biomarker.
Molecular and Cellular Biology, 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
Pawel Jaruga - One of the best experts on this subject based on the ideXlab platform.
-
Recognition of DNA adducts by edited and unedited forms of DNA glycosylase NEIL1.
DNA repair, 2019Co-Authors: Irina G Minko, Sheila S David, Pawel Jaruga, Michael P Stone, Jongchan Yeo, Vladimir Vartanian, Naoto N. Tozaki, Erdem Coskun, Sanem Hosbas Coskun, Martin EgliAbstract:Abstract Pre-mRNA encoding human NEIL1 undergoes editing by adenosine deaminase ADAR1 that converts a single adenosine to inosine, and this conversion results in an amino acid change of lysine 242 to arginine. Previous investigations of the catalytic efficiencies of the two forms of the enzyme revealed differential release of thymine glycol (ThyGly) from synthetic oligodeoxynucleotides, with the unedited form, NEIL1 K242 being ≈30-fold more efficient than the edited NEIL1 K242R. In contrast, when these enzymes were reacted with oligodeoxynucleotides containing guanidinohydantoin or spiroiminohydantoin, the edited K242R form was ≈3-fold more efficient than the unedited NEIL1. However, no prior studies have investigated the efficiencies of these two forms of NEIL1 on either high-molecular weight DNA containing multiple oxidatively-induced base damages, or oligodeoxynucleotides containing a bulky alkylated formamidopyrimidine. To understand the extent of changes in substrate recognition, γ-irradiated calf thymus DNA was treated with either edited or unedited NEIL1 and the released DNA base lesions analyzed by gas chromatography-tandem mass spectrometry. Of all the measured DNA lesions, imidazole ring-opened 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were preferentially released by both NEIL1 enzymes with K242R being ≈1.3 and 1.2-fold more efficient than K242 on excision of FapyAde and FapyGua, respectively. Consistent with the prior literature, large differences (≈7.5 to 12-fold) were measured in the excision of ThyGly from genomic DNA by the unedited versus edited NEIL1. In contrast, the edited NEIL1 was more efficient (≈3 to 5-fold) on release of 5-hydroxy-cytosine. Excision kinetics on DNA containing a site-specific aflatoxin B1-FapyGua adduct revealed an ≈1.4-fold higher rate by the unedited NEIL1. Molecular modeling provides insight into these differential substrate specificities. The results of this study and in particular, the comparison of substrate specificities of unedited and edited NEIL1 using biologically and clinically important base lesions, are critical for defining its role in preservation of genomic integrity.
-
Enhanced sensitivity of NEIL1-/- mice to chronic UVB exposure.
DNA repair, 2016Co-Authors: Marcus J. Calkins, Amanda K Mccullough, Pawel Jaruga, Miral Dizdaroglu, Güldal Kirkali, Vladimir Vartanian, Nichole Owen, R. Stephen LloydAbstract:Abstract Oxidative stress and reactive oxygen species (ROS)-induced DNA base damage are thought to be central mediators of UV-induced carcinogenesis and skin aging. However, increased steady-state levels of ROS-induced DNA base damage have not been reported after chronic UV exposure. Accumulation of ROS-induced DNA base damage is governed by rates of lesion formation and repair. Repair is generally performed by Base Excision Repair (BER), which is initiated by DNA glycosylases, such as 8-oxoguanine glycosylase and Nei-Endonuclease VIII-Like 1 (NEIL1). In the current study, UV light (UVB) was used to elicit protracted low-level ROS challenge in wild-type (WT) and NEIL1 −/− mouse skin. Relative to WT controls, NEIL1 −/− mice showed an increased sensitivity to tissue destruction from the chronic UVB exposure, and corresponding enhanced chronic inflammatory responses as measured by cytokine message levels and profiling, as well as neutrophil infiltration. Additionally, levels of several ROS-induced DNA lesions were measured including 4,6-diamino-5-formamidopyrimidine (FapyGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyAde), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxyuracil (5,6-diOH-Ura) and thymine glycol (ThyGly). In WT mice, chronic UVB exposure led to increased steady-state levels of FapyGua, FapyAde, and ThyGly with no significant increases in 8-OH-Gua or 5,6-diOH-Ura. Interestingly, the lesions that accumulated were all substrates of NEIL1. Collectively, these data suggest that NEIL1-initiated repair of a subset of ROS-induced DNA base lesions may be insufficient to prevent the initiation of inflammatory pathways during chronic UV exposure in mouse skin.
-
abstract 2421 small molecule inhibitors of dna glycosylases as potential drugs in cancer therapy
Cancer Research, 2014Co-Authors: Miral Dizdaroglu, Pawel JarugaAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA glycosylases are required to initiate the DNA base excision repair pathway of lesions that are formed by DNA damage. Recently, several DNA glycosylases including NEIL1, OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies for cancer treatment. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. In this work, we designed experimental strategies to discover small molecule inhibitors of NEIL1, with subsequent analyses of the specificity of those molecules for other DNA glycosylases. Using NEIL1 as the proof-of-principle DNA glycosylase, we developed a fluorescence-based assay that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of purine-derived lesions. We also applied gas chromatography/isotope-dilution tandem mass spectrometry for the measurement of the effect of preferred small molecule inhibitors on activities of the DNA glycosylases NEIL1, OGG1 and NTH1. The release of modified bases, which are known substrates of these enzymes, was measured in the presence and absence of the inhibitors. These data revealed that several of the purine analogs were general glycosylase inhibitors with one compound being the most effective inhibitor for all three enzymes. However, there were significance differences in inhibition of enzymatic activities among these three DNA glycosylases. Overall, this work forms the foundation for the future discovery of DNA repair inhibitors as drugs in cancer therapy for the entire family of DNA glycosylases. Citation Format: Miral Dizdaroglu, Pawel Jaruga. Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2014-2421
-
abstract 2421 small molecule inhibitors of dna glycosylases as potential drugs in cancer therapy
Cancer Research, 2014Co-Authors: Miral Dizdaroglu, Pawel JarugaAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA glycosylases are required to initiate the DNA base excision repair pathway of lesions that are formed by DNA damage. Recently, several DNA glycosylases including NEIL1, OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies for cancer treatment. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. In this work, we designed experimental strategies to discover small molecule inhibitors of NEIL1, with subsequent analyses of the specificity of those molecules for other DNA glycosylases. Using NEIL1 as the proof-of-principle DNA glycosylase, we developed a fluorescence-based assay that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of purine-derived lesions. We also applied gas chromatography/isotope-dilution tandem mass spectrometry for the measurement of the effect of preferred small molecule inhibitors on activities of the DNA glycosylases NEIL1, OGG1 and NTH1. The release of modified bases, which are known substrates of these enzymes, was measured in the presence and absence of the inhibitors. These data revealed that several of the purine analogs were general glycosylase inhibitors with one compound being the most effective inhibitor for all three enzymes. However, there were significance differences in inhibition of enzymatic activities among these three DNA glycosylases. Overall, this work forms the foundation for the future discovery of DNA repair inhibitors as drugs in cancer therapy for the entire family of DNA glycosylases. Citation Format: Miral Dizdaroglu, Pawel Jaruga. Small molecule inhibitors of DNA glycosylases as potential drugs in cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2421. doi:10.1158/1538-7445.AM2014-2421
-
Inhibition of DNA glycosylases via small molecule purine analogs.
PLOS ONE, 2013Co-Authors: Aaron C. Jacobs, Marcus J. Calkins, Amanda K Mccullough, Dorjbal Dorjsuren, Anton Simeonov, Ajit Jadhav, David G Maloney, Pawel Jaruga, Miral Dizdaroglu, R. Stephen LloydAbstract:Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.
Tapas K Hazra - One of the best experts on this subject based on the ideXlab platform.
-
the c terminal domain ctd of human dna glycosylase NEIL1 is required for forming berosome repair complex with dna replication proteins at the replicating genome dominant negative function of the ctd
Journal of Biological Chemistry, 2015Co-Authors: Pavana M Hegde, Alan E Tomkinson, Istvan Boldogh, Guo Min Li, Shiladitya Sengupta, Joy Mitra, Arijit Dutta, Sanjay Adhikari, Tapas K HazraAbstract:The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.
-
prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 dna glycosylase together with replication proteins
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Muralidhar L Hegde, Alan E Tomkinson, Istvan Boldogh, Tapas K Hazra, Pavana M Hegde, Larry J Bellot, Santi M Mandal, Guo Min Li, Sankar MitraAbstract:Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.
-
Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction.
The Journal of biological chemistry, 2012Co-Authors: Muralidhar L Hegde, Istvan Boldogh, Tapas K Hazra, Pavana M Hegde, Larry J Bellot, Srijita Banerjee, Sankar MitraAbstract:Abstract Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (Kd = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.
-
induction of NEIL1 and neil2 dna glycosylases in aniline induced splenic toxicity
Toxicology and Applied Pharmacology, 2011Co-Authors: Jianling Wang, Tapas K Hazra, Sherif Z Abdelrahman, Paul J Boor, Firoze M KhanAbstract:The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.
-
RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.
DNA repair, 2010Co-Authors: Corey A Theriot, Muralidhar L Hegde, Tapas K Hazra, Sankar MitraAbstract:The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome.
Dmitry O. Zharkov - One of the best experts on this subject based on the ideXlab platform.
-
requirements for dna bubble structure for efficient cleavage by helix two turn helix dna glycosylases
Mutagenesis, 2020Co-Authors: Kristina A Makasheva, Anton V Endutkin, Dmitry O. ZharkovAbstract:: Oxidative DNA lesions, constantly generated by both endogenous and environmentally induced reactive oxygen species, are removed via the base excision repair pathway. In bacteria, Fpg and Nei DNA glycosylases, belonging to the helix-two-turn-helix (H2TH) structural superfamily, remove oxidised purines and pyrimidines, respectively. Interestingly, the human H2TH family glycosylases, NEIL1, NEIL2 and NEIL3, have been reported to prefer oxidative lesions in DNA bubbles or single-stranded DNA. It had been hypothesised that NEIL2 might be involved in the repair of lesions in transcription bubbles; however, bubble-like structures may appear in other cellular contexts such as displacement loops (D-loops) associated with transcription, recombination or telomere maintenance. The activities of bacterial Fpg and Nei on bubble substrates were not addressed. Also, it is not known whether H2TH enzymes process bubbles containing the third DNA or RNA strand, and how the bubble length and position of the lesion within a bubble affect the excision. We have investigated the removal of 8-oxoguanine (8-oxoG) and 5,6-dihydrouracil (DHU) by Escherichia coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different numbers of unpaired bases (6-30), bubbles containing the lesion in different positions and D-loops with the third strand made of DNA or RNA. Fpg, NEIL1 and NEIL2 efficiently excised lesions located within bubbles, with NEIL1 and NEIL2 being specific for DHU, and Fpg removing both 8-oxoG and DHU. Nei, in contrast, was significantly active only on DHU located in double-stranded DNA. Fpg and NEIL1 also tolerated the presence of the third strand of either DNA or RNA in D-loops if the lesion was in the single-stranded part, and Fpg, Nei and NEIL1 excised lesions from the double-stranded DNA part of D-loops. The presence of an additional unpaired 5'-tail of DNA or RNA did not affect the activity. No significant position preference for lesions in a 12-mer bubble was found. Overall, the activities of Fpg, NEIL1 and NEIL2 on these non-canonical substrates are consistent with the possibility that these enzymes may participate in the repair in structures arising during transcription or homologous recombination.
-
Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1.
Journal of molecular biology, 2019Co-Authors: Olga A. Kladova, Inga R Grin, Olga S. Fedorova, Nikita A. Kuznetsov, Dmitry O. ZharkovAbstract:Abstract Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix–two turn–helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine–DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Forster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.
-
effect of the multifunctional proteins rpa yb 1 and xpc repair factor on ap site cleavage by dna glycosylase NEIL1
Journal of Molecular Recognition, 2012Co-Authors: P E Pestryakov, Inga R Grin, Elizaveta E Fomina, I O Petruseva, Irina A. Eliseeva, Loic Hamon, Dmitry O. Zharkov, Patrick A CurmiAbstract:DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1. Copyright © 2012 John Wiley & Sons, Ltd.
-
Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1
Journal of Molecular Recognition, 2012Co-Authors: P E Pestryakov, Inga R Grin, I O Petruseva, Irina A. Eliseeva, Patrick A Curmi, Loic Hamon, Dmitry O. Zharkov, Elizaveta Fomina, Ekaterina Kim, Lev OvchinnikovAbstract:DNA glycosylases are key enzymes in thefirst step of base excision DNA repair, recognizing DNA damage and catalyzingthe release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP)lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at thefirst stepof repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 andRPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site waslocated in a 60nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP sitewas located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably aconsequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation groupC protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but hadno effect on AP site cleavage by NEIL1.
-
heavy metal ions affect the activity of dna glycosylases of the fpg family
Biochemistry, 2009Co-Authors: Inga R Grin, Dmitry O. Zharkov, P G Konorovsky, G A NevinskyAbstract:Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K m = 150 nM and k cat = 1.2 min−1, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al3+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+, and Fe2+ in Tris-HCl buffer and by Cd2+, Zn2+, Cu2+, and Fe2+ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I50 for NEIL1 inhibition were 7 µM for Cd2+, 16 µM for Zn2+, and 400 µM for Cu2+. The inhibition of NEIL1 by Cd2+, Zn2+, and Cu2+ was at least partly due to the formation of metal-DNA complexes. In the case of Cd2+ and Cu2+, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.
