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Sankar Mitra - One of the best experts on this subject based on the ideXlab platform.
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Increased human AP Endonuclease 1 level confers protection against the paternal age effect in mice.
Mutation research, 2015Co-Authors: Jamila R. Sanchez, Tadahide Izumi, Sankar Mitra, Traci L. Reddick, Marissa Perez, Victoria E. Centonze, C. Alex Mcmahan, Christi A. WalterAbstract:Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency APpears to be associated with a decrease in the DNA repair protein, AP Endonuclease 1 (APEX1) and APex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased APoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.
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dual regulatory roles of human AP Endonuclease APe1 ref 1 in cdkn1a p21 expression
PLOS ONE, 2013Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. BhakatAbstract:The human AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53’s DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1’s stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1’s negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1’s another binding site in p21’s proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4’s association with APE1, their binding to the promoter and p21 repression. These results together establish APE1’s role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.
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Dual Regulatory Roles of Human AP-Endonuclease (APE1/Ref-1) in CDKN1A/p21 Expression
PloS one, 2013Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. BhakatAbstract:The human AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53’s DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1’s stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1’s negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1’s another binding site in p21’s proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4’s association with APE1, their binding to the promoter and p21 repression. These results together establish APE1’s role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.
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MD simulation and experimental evidence for Mg²+ binding at the B site in human AP Endonuclease 1.
Bioinformation, 2011Co-Authors: Numan Oezguen, Anil K Mantha, Tadahide Izumi, Catherine H Schein, Sankar Mitra, Werner BraunAbstract:APurinic/APyrimidinic Endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg2+ dependent reaction. Despite characterization of nine X-ray crystallogrAPhic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg2+ binding at the “A” site, we show experimentally that this mutant still requires Mg2+ at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg2+ placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg2+ at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg2+ binding at the B site is involved in the reaction mechanism associated with Endonuclease function of APE1. Abbreviations APE - AP-Endonuclease, AP site - APurinic/APyrimidinic site, BER - base excision repair, Ref-1 - redox factor 1, hAPE1 - human APE1, WT - wild type, MD - molecular dynamics, THF - tetrahydrofuran.
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md simulation and experimental evidence for mg binding at the b site in human AP Endonuclease 1
Bioinformation, 2011Co-Authors: Numan Oezguen, Anil K Mantha, Tadahide Izumi, Catherine H Schein, Sankar Mitra, Werner BraunAbstract:APurinic/APyrimidinic Endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg2+ dependent reaction. Despite characterization of nine X-ray crystallogrAPhic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg2+ binding at the “A” site, we show experimentally that this mutant still requires Mg2+ at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg2+ placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg2+ at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg2+ binding at the B site is involved in the reaction mechanism associated with Endonuclease function of APE1. Abbreviations APE - AP-Endonuclease, AP site - APurinic/APyrimidinic site, BER - base excision repair, Ref-1 - redox factor 1, hAPE1 - human APE1, WT - wild type, MD - molecular dynamics, THF - tetrahydrofuran.
Kishor K. Bhakat - One of the best experts on this subject based on the ideXlab platform.
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Dual Regulatory Roles of Human AP-Endonuclease (APE1/Ref-1) in CDKN1A/p21 Expression
PloS one, 2013Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. BhakatAbstract:The human AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53’s DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1’s stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1’s negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1’s another binding site in p21’s proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4’s association with APE1, their binding to the promoter and p21 repression. These results together establish APE1’s role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.
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dual regulatory roles of human AP Endonuclease APe1 ref 1 in cdkn1a p21 expression
PLOS ONE, 2013Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. BhakatAbstract:The human AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53’s DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1’s stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53-null HCT116 cells. Ectopic expression of p53 in the p53-null cells abrogated this repression suggesting that APE1’s negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1’s another binding site in p21’s proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53-null cells inhibited AP4’s association with APE1, their binding to the promoter and p21 repression. These results together establish APE1’s role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.
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transcriptional regulatory functions of mammalian AP Endonuclease APe1 ref 1 an essential multifunctional protein
Antioxidants & Redox Signaling, 2009Co-Authors: Kishor K. Bhakat, Anil K Mantha, Sankar MitraAbstract:Abstract The mammalian AP-Endonuclease (APE1/Ref-1) plays a central role in the repair of oxidized and alkylated bases in mammalian genomes via the base excision repair (BER) pathway. However, APE1, unlike its E. coli prototype Xth, has two unique and APparently distinct transcriptional regulatory activities. APE1 functions as a redox effector factor (Ref-1) for several transcription factors including AP-1, HIF1-α, and p53. APE1 was also identified as a direct trans-acting factor for repressing human parathyroid hormone (PTH) and renin genes by binding to the negative calcium-response element (nCaRE) in their promoters. We have characterized APE1's post-translational modification, namely, acetylation which modulates its transcriptional regulatory function. Furthermore, stable interaction of APE1 with several other trans-acting factors including HIF-1α, STAT3, YB-1, HDAC1, and CBP/p300 and formation of distinct trans-acting complexes support APE1's direct regulatory function for diverse genes. Multiple fun...
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Transcriptional regulatory functions of mammalian AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein
Antioxidants & redox signaling, 2009Co-Authors: Kishor K. Bhakat, Anil K Mantha, Sankar MitraAbstract:Abstract The mammalian AP-Endonuclease (APE1/Ref-1) plays a central role in the repair of oxidized and alkylated bases in mammalian genomes via the base excision repair (BER) pathway. However, APE1, unlike its E. coli prototype Xth, has two unique and APparently distinct transcriptional regulatory activities. APE1 functions as a redox effector factor (Ref-1) for several transcription factors including AP-1, HIF1-α, and p53. APE1 was also identified as a direct trans-acting factor for repressing human parathyroid hormone (PTH) and renin genes by binding to the negative calcium-response element (nCaRE) in their promoters. We have characterized APE1's post-translational modification, namely, acetylation which modulates its transcriptional regulatory function. Furthermore, stable interaction of APE1 with several other trans-acting factors including HIF-1α, STAT3, YB-1, HDAC1, and CBP/p300 and formation of distinct trans-acting complexes support APE1's direct regulatory function for diverse genes. Multiple fun...
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regulation of the human AP Endonuclease APe1 ref 1 expression by the tumor suppressor p53 in response to dna damage
Nucleic Acids Research, 2008Co-Authors: Ahmad Zaky, Ranajoy Chattopadhyay, Ahmad Bassiouny, Carlos Busso, Tadahide Izumi, Sankar Mitra, Kishor K. BhakatAbstract:The human AP-Endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-Endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of APoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of APoptosis in response to DNA damage.
Olga S Fedorova - One of the best experts on this subject based on the ideXlab platform.
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Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1.
Acta naturae, 2020Co-Authors: Alexandra A Kuznetsova, Olga S Fedorova, D. S. Novopashina, Nikita A. KuznetsovAbstract:Human APurinic/APyrimidinic (AP) Endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5′-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine–purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.
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Role of Ionizing Amino Acid Residues in the Process of DNA Binding by Human AP Endonuclease 1 and in Its Catalysis
The journal of physical chemistry. B, 2019Co-Authors: Irina V. Alekseeva, Olga S Fedorova, Artemiy S. Bakman, Yury N. Vorobjev, Nikita A. KuznetsovAbstract:In the repair of the damage to bases, human APurinic/APyrimidinic (AP) Endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme’s active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pKa of His-309 located in the active site...
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Kinetic Features of 3'-5' Exonuclease Activity of Human AP-Endonuclease APE1.
Molecules (Basel Switzerland), 2018Co-Authors: Alexandra A Kuznetsova, Olga S Fedorova, Nikita A. KuznetsovAbstract:Human APurinic/APyrimidinic (AP)-Endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5′ to the damage. In addition to the AP-Endonuclease activity, APE1 possesses 3′-5′ exonuclease activity, which presumably is responsible for cleaning up nonconventional 3′ ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3′-end nucleotide removal in the 3′-5′ exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5′ dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3′ end of the short oligonucleotide. The impact of the 3′-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3′-5′ exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3′ nucleotide removal by APE1 in the 3′-5′ exonuclease process is the release of the detached nucleotide from the enzyme’s active site.
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Kinetic Features of 3′-5′ Exonuclease Activity of Human AP-Endonuclease APE1
MDPI AG, 2018Co-Authors: Alexandra A Kuznetsova, Olga S Fedorova, Nikita A. KuznetsovAbstract:Human APurinic/APyrimidinic (AP)-Endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5′ to the damage. In addition to the AP-Endonuclease activity, APE1 possesses 3′-5′ exonuclease activity, which presumably is responsible for cleaning up nonconventional 3′ ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3′-end nucleotide removal in the 3′-5′ exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5′ dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3′ end of the short oligonucleotide. The impact of the 3′-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3′-5′ exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3′ nucleotide removal by APE1 in the 3′-5′ exonuclease process is the release of the detached nucleotide from the enzyme’s active site
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pre steady state fluorescence analysis of damaged dna transfer from human dna glycosylases to AP Endonuclease APe1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Murat Saparbaev, Nikita A. Kuznetsov, Alexander A. Ishchenko, Olga S FedorovaAbstract:Abstract Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (APurinic/APyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-Endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP Endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
Alexander A. Ishchenko - One of the best experts on this subject based on the ideXlab platform.
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Differential drug sensitivity of AP Endonuclease—deficient E. coli strain BH110 (DE3) carrying a plasmid coding for the H. pylori AP Endonuclease.
2018Co-Authors: Aigerim Turgimbayeva, Murat Saparbaev, Sailau Abeldenov, Dmitry O. Zharkov, Yerla Ramankulov, Alexander A. Ishchenko, Ekbola KhassenovAbstract:The strains are represented as follows: the BH110 (DE3) strain carrying control empty vector pBluescript II SK+ (pBSK) (□), strain BH110 (DE3) carrying pBW21-Nfo (■), or pBSK-HpXth (▼), or pBSK-HpXth-D144N (△). Each survival curve represents at least three independent experiments. (A) The survival of the MMS-treated E. coli AP Endonuclease—deficient strains. (B) The survival of the E. coli AP Endonuclease—deficient strains under oxidative stress. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details, see Materials and methods.
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Phylogenetic analysis and structural models of H. pylori AP Endonuclease.
2018Co-Authors: Aigerim Turgimbayeva, Murat Saparbaev, Sailau Abeldenov, Dmitry O. Zharkov, Yerla Ramankulov, Alexander A. Ishchenko, Bekbolat KhassenovAbstract:(A) An unrooted phylogenetic tree of the EEP superfamily AP Endonucleases (230 sequences, one species per taxonomic class). Different families are indicated by colored arcs: yellow, ExoIII-like; green, NAPe-like; blue, Mth212-like; magenta, APe1-like; and red, APe2-like. The clade combining HpXth, B. subtilis ExoA, and the related bacterial and archaeal sequences is labeled “Archaea+Bacteria.” The known NIR-proficient and NIR-deficient enzymes are indicated. (B) Overlay of the structure of human APE1 (1DEW, cyan) and the HpXth model (green). All active-site residues are shown but left unlabeled for clarity. The DNA groove—contacting residues possibly involved in the NIR function are indicated by red carbon atoms. (C) Alignment of HpXth and four core members of AP Endonuclease families (human APE1 catalytic domain, E. coli Xth, N. meningitidis NAPe, and M. thermautotrophicus Mth212). Metal-binding sites A and B are red and blue, respectively, non—metal-binding active site residues are green; other DNA-binding residues are yellow.
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pre steady state fluorescence analysis of damaged dna transfer from human dna glycosylases to AP Endonuclease APe1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Murat Saparbaev, Nikita A. Kuznetsov, Alexander A. Ishchenko, Olga S FedorovaAbstract:Abstract Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (APurinic/APyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-Endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP Endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
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pre steady state fluorescence analysis of damaged dna transfer from human dna glycosylases to AP Endonuclease APe1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Murat Saparbaev, Nikita A. Kuznetsov, Alexander A. Ishchenko, Olga S FedorovaAbstract:Abstract Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (APurinic/APyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-Endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP Endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
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genetic and biochemical characterization of human AP Endonuclease 1 mutants deficient in nucleotide incision repair activity
PLOS ONE, 2010Co-Authors: Aurore Gelin, Olga S Fedorova, Murat Saparbaev, Modesto Redrejorodriguez, Jacques Laval, Alexander A. IshchenkoAbstract:Background Human APurinic/APyrimidinic Endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells. Methodology/Principal Finding We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were cAPable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP Endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flAP Endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells. Conclusion/Significance Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.
Murat Saparbaev - One of the best experts on this subject based on the ideXlab platform.
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Differential drug sensitivity of AP Endonuclease—deficient E. coli strain BH110 (DE3) carrying a plasmid coding for the H. pylori AP Endonuclease.
2018Co-Authors: Aigerim Turgimbayeva, Murat Saparbaev, Sailau Abeldenov, Dmitry O. Zharkov, Yerla Ramankulov, Alexander A. Ishchenko, Ekbola KhassenovAbstract:The strains are represented as follows: the BH110 (DE3) strain carrying control empty vector pBluescript II SK+ (pBSK) (□), strain BH110 (DE3) carrying pBW21-Nfo (■), or pBSK-HpXth (▼), or pBSK-HpXth-D144N (△). Each survival curve represents at least three independent experiments. (A) The survival of the MMS-treated E. coli AP Endonuclease—deficient strains. (B) The survival of the E. coli AP Endonuclease—deficient strains under oxidative stress. The statistical significance of the differences among the mean values were evaluated using two-tailed Student’s test (***P < 0.001; **P < 0.01; *P < 0.05 and nsP > 0.05). For details, see Materials and methods.
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Phylogenetic analysis and structural models of H. pylori AP Endonuclease.
2018Co-Authors: Aigerim Turgimbayeva, Murat Saparbaev, Sailau Abeldenov, Dmitry O. Zharkov, Yerla Ramankulov, Alexander A. Ishchenko, Bekbolat KhassenovAbstract:(A) An unrooted phylogenetic tree of the EEP superfamily AP Endonucleases (230 sequences, one species per taxonomic class). Different families are indicated by colored arcs: yellow, ExoIII-like; green, NAPe-like; blue, Mth212-like; magenta, APe1-like; and red, APe2-like. The clade combining HpXth, B. subtilis ExoA, and the related bacterial and archaeal sequences is labeled “Archaea+Bacteria.” The known NIR-proficient and NIR-deficient enzymes are indicated. (B) Overlay of the structure of human APE1 (1DEW, cyan) and the HpXth model (green). All active-site residues are shown but left unlabeled for clarity. The DNA groove—contacting residues possibly involved in the NIR function are indicated by red carbon atoms. (C) Alignment of HpXth and four core members of AP Endonuclease families (human APE1 catalytic domain, E. coli Xth, N. meningitidis NAPe, and M. thermautotrophicus Mth212). Metal-binding sites A and B are red and blue, respectively, non—metal-binding active site residues are green; other DNA-binding residues are yellow.
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pre steady state fluorescence analysis of damaged dna transfer from human dna glycosylases to AP Endonuclease APe1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Murat Saparbaev, Nikita A. Kuznetsov, Alexander A. Ishchenko, Olga S FedorovaAbstract:Abstract Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (APurinic/APyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-Endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP Endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
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pre steady state fluorescence analysis of damaged dna transfer from human dna glycosylases to AP Endonuclease APe1
Biochimica et Biophysica Acta, 2014Co-Authors: Alexandra A Kuznetsova, Murat Saparbaev, Nikita A. Kuznetsov, Alexander A. Ishchenko, Olga S FedorovaAbstract:Abstract Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (APurinic/APyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-Endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP Endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.
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genetic and biochemical characterization of human AP Endonuclease 1 mutants deficient in nucleotide incision repair activity
PLOS ONE, 2010Co-Authors: Aurore Gelin, Olga S Fedorova, Murat Saparbaev, Modesto Redrejorodriguez, Jacques Laval, Alexander A. IshchenkoAbstract:Background Human APurinic/APyrimidinic Endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells. Methodology/Principal Finding We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were cAPable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP Endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flAP Endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells. Conclusion/Significance Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.