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AP Endonuclease

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Sankar Mitra – One of the best experts on this subject based on the ideXlab platform.

  • Increased human AP Endonuclease 1 level confers protection against the paternal age effect in mice.
    Mutation research, 2015
    Co-Authors: Jamila R. Sanchez, Sankar Mitra, Tadahide Izumi, Traci L. Reddick, Marissa Perez, Victoria E. Centonze, C. Alex Mcmahan, Christi A. Walter

    Abstract:

    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.

  • Dual Regulatory Roles of Human APEndonuclease (APE1/Ref-1) in CDKN1A/p21 Expression
    PloS one, 2013
    Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. Bhakat

    Abstract:

    The human APEndonuclease (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.

  • dual regulatory roles of human AP Endonuclease APe1 ref 1 in cdkn1a p21 expression
    PLOS ONE, 2013
    Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. Bhakat

    Abstract:

    The human APEndonuclease (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.

Kishor K. Bhakat – One of the best experts on this subject based on the ideXlab platform.

  • Dual Regulatory Roles of Human APEndonuclease (APE1/Ref-1) in CDKN1A/p21 Expression
    PloS one, 2013
    Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. Bhakat

    Abstract:

    The human APEndonuclease (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.

  • dual regulatory roles of human AP Endonuclease APe1 ref 1 in cdkn1a p21 expression
    PLOS ONE, 2013
    Co-Authors: Shiladitya Sengupta, Sankar Mitra, Kishor K. Bhakat

    Abstract:

    The human APEndonuclease (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.

  • transcriptional regulatory functions of mammalian AP Endonuclease APe1 ref 1 an essential multifunctional protein
    Antioxidants & Redox Signaling, 2009
    Co-Authors: Kishor K. Bhakat, Anil K Mantha, Sankar Mitra

    Abstract:

    Abstract The mammalian APEndonuclease (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…

Olga S Fedorova – One of the best experts on this subject based on the ideXlab platform.

  • Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1.
    Acta naturae, 2020
    Co-Authors: Alexandra A Kuznetsova, Olga S Fedorova, D. S. Novopashina, Nikita A. Kuznetsov

    Abstract:

    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.

  • 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, 2019
    Co-Authors: Irina V. Alekseeva, Olga S Fedorova, Artemiy S. Bakman, Yury N. Vorobjev, Nikita A. Kuznetsov

    Abstract:

    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…

  • Kinetic Features of 3′-5′ Exonuclease Activity of Human APEndonuclease APE1.
    Molecules (Basel Switzerland), 2018
    Co-Authors: Alexandra A Kuznetsova, Olga S Fedorova, Nikita A. Kuznetsov

    Abstract:

    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 APEndonuclease 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.