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A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease

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Abstract

Mammalian apurinic/apyrimidinic endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are highly toxic, mutagenic, and implicated in carcinogenesis. Also, reducing the activity of APE1 protein in cancer cells and tumors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. In general, mouse models are used in studies of basic mechanisms of carcinogenesis, as well as pre-clinical studies before transitioning into humans. Human APE1 (hAPE1) has previously been cloned, expressed, and extensively characterized. However, the knowledge regarding the characterization of mouse APE1 (mAPE1) is very limited. Here we have expressed and purified full-length hAPE1 and mAPE1 in and from E. coli to near homogeneity. mAPE1 showed comparable fast reaction kinetics to its human counterpart. Steady-state enzyme kinetics showed an apparent K m of 91 nM and k cat of 4.2 s−1 of mAPE1 for the THF cleavage reaction. For hAPE1 apparent K m and k cat were 82 nM and 3.2 s−1, respectively, under similar reaction conditions. However, k cat/K m were in similar range for both APE1s. The optimum pH was in the range of 7.5–8 for both APE1s and had an optimal activity at 50–100 mM KCl, and they showed Mg2+ dependence and abrogation of activity at high salt. Circular dichroism spectroscopy revealed that increasing the Mg2+ concentration altered the ratio of “turns” to “β-strands” for both proteins, and this change may be associated with the conformational changes required to achieve an active state. Overall, compared to hAPE1, mAPE1 has higher K m and k cat values. However, overall results from this study suggest that human and mouse APE1s have mostly similar biochemical and biophysical properties. Thus, the conclusions of mouse studies to elucidate APE1 biology and its role in carcinogenesis may be extrapolated to apply to human biology. This includes the development and validation of effective APE1 inhibitors as chemosensitizers in clinical studies.

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Abbreviations

BER:

Base excision repair

AP:

Apurinic/apyrimidinic

mAPE1:

Mouse apurinic/apyrimidinic endonuclease 1

hAPE1:

Human apurinic/apyrimidinic endonuclease 1

THF:

Tetrahydrofuran

References

  1. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715

    Article  PubMed  CAS  Google Scholar 

  2. Clancy S (2008) DNA damage & repair: mechanisms for maintaining DNA integrity. Nat Educ 1:1

    Google Scholar 

  3. Larson K, Sahm J, Shenkar R, Strauss B (1985) Methylation-induced blocks to in vitro DNA replication. Mutat Res 150:77–84

    Article  PubMed  CAS  Google Scholar 

  4. Lindahl T (1982) DNA repair enzymes. Annu Rev Biochem 51:61–87

    Article  PubMed  CAS  Google Scholar 

  5. Barnes T, Kim WC, Mantha AK, Kim SE, Izumi T, Mitra S, Lee CH (2009) Identification of apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA. Nucleic Acids Res 37:3946–3958

    Article  PubMed  CAS  Google Scholar 

  6. Chattopadhyay R, Das S, Maiti AK, Boldogh I, Xie J, Hazra TK, Kohno K, Mitra S, Bhakat KK (2008) Regulatory role of human AP-endonuclease (APE1/REF-1) in YB-1-mediated activation of the multidrug resistance gene MDR1. Mol Cell Biol 28:7066–7080

    Article  PubMed  CAS  Google Scholar 

  7. Bhakat KK, Mantha AK, Mitra S (2009) Transcriptional regulatory functions of mammalian AP-endonuclease (APE11/Ref-1), an essential multifunctional protein. Antioxid Redox Signal 11:621–638

    Article  PubMed  CAS  Google Scholar 

  8. Vascotto C, Fantini D, Romanello M, Cesaratto L, Deganuto M, Leonardi A, Radicella JP, Kelley MR, D’Ambrosio C, Scaloni A, Quadrifoglio F, Tell G (2009) APE1/Ref-1 interacts with NPM1 within nucleoli and plays a role in the rRNA quality control process. Mol Cell Biol 29:1834–1854

    Article  PubMed  CAS  Google Scholar 

  9. McNeill DR, Lam W, DeWeese TL, Cheng YC, Wilson DM III (2009) Impairment of APE11 function enhances cellular sensitivity to clinically relevant alkylators and antimetabolites. Mol Cancer Res 7:897–906

    Article  PubMed  CAS  Google Scholar 

  10. Liu L, Gerson SL (2004) Therapeutic impact of methoxyamine: blocking repair of abasic sites in the base excision repair pathway. Curr Opin Investig Drugs 5:623–627

    PubMed  CAS  Google Scholar 

  11. Taverna P, Liu L, Hwang HS, Hanson AJ, Kinsella TJ, Gerson SL (2001) Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells. Mutat Res 485:269–281

    PubMed  CAS  Google Scholar 

  12. Liu L, Nakatsuru Y, Gerson SL (2002) Base excision repair as a therapeutic target in colon cancer. Clin Cancer Res 8:2985–2991

    PubMed  CAS  Google Scholar 

  13. Madhusudan S, Hickson ID (2005) DNA repair inhibition: a selective tumour targeting strategy. Trends Mol Med 11:503–511

    Article  PubMed  CAS  Google Scholar 

  14. Lou M, Kelly MR (2004) Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone. Anticancer Res 24:2127–2134

    Google Scholar 

  15. Madhusudan S, Smart F, Shrimpton P, Parsons JL, Gardiner L, Houlbrook S, Talbot DC, Hammonds T, Freemont PA, Sternberg MJ, Dianov GL, Hickson ID (2005) Isolation of a small molecule inhibitor of DNA base excision repair. Nucleic Acids Res 33:4711–4724

    Article  PubMed  CAS  Google Scholar 

  16. Simeonov A, Kulkarni A, Dorjsuren D, Jadhav A, Shen M, McNeill DR, Austin CP, Wilson DM 3rd (2009) Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1. PLoS One 4:e5740

    Article  PubMed  Google Scholar 

  17. Damia G, D’Incalci M (2007) Targeting DNA repair as a promising approach in cancer therapy. Eur J Cancer 43:1791–1801

    Article  PubMed  CAS  Google Scholar 

  18. Izumi T, Brown DB, Naidu CV, Bhakat KK, Macinnes MA, Saito H, Chen DJ, Mitra S (2005) Two essential but distinct functions of the mammalian abasic endonuclease. Proc Natl Acad Sci USA 102:5739–5743

    Article  PubMed  CAS  Google Scholar 

  19. Xanthoudakis S, Smeyne RJ, Wallace JD, Curran T (1996) The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proc Natl Acad Sci USA 93:8919–8923

    Article  PubMed  CAS  Google Scholar 

  20. Meira LB, Devaraj S, Kisby GE, Burns DK, Daniel RL, Hammer RE, Grundy S, Jialal I, Friedberg EC (2001) Heterozygosity for the mouse Apex gene results in phenotypes associated with oxidative stress. Cancer Res 61:5552–5557

    PubMed  CAS  Google Scholar 

  21. Mantha AK, Oezguen N, Bhakat KK, Izumi T, Braun W, Mitra S (2008) Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1. J Mol Biol 379:28–37

    Article  PubMed  CAS  Google Scholar 

  22. Adhikari S, Manthena PV, Sajwan K, Kota KK, Roy R (2010) A unified method for purification of basic proteins. Anal Biochem 400:203–206

    Article  PubMed  CAS  Google Scholar 

  23. Adhikari S, Toretsky JA, Yuan L, Roy R (2006) Magnesium, essential for base excision repair enzymes, inhibits substrate binding of N-methylpurine-DNA glycosylase. J Biol Chem 281:29525–29532

    Article  PubMed  CAS  Google Scholar 

  24. Compton LA, Johnson WC Jr (1986) Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 155:155–167

    Article  PubMed  CAS  Google Scholar 

  25. Manavalan P, Johnson WC Jr (1987) Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Anal Biochem 167:76–85

    Article  PubMed  CAS  Google Scholar 

  26. Sreerama N, Woody RW (2000) Estimation of protein secondary structure from CD spectra: comparison of CONTIN, SELCON and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260

    Article  PubMed  CAS  Google Scholar 

  27. Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:668–673

    Article  Google Scholar 

  28. Adhikari S, Choudhury S, Mitra PS, Dubash JJ, Sajankila SP, Roy R (2008) Targeting base excision repair for chemosensitization. Anticancer Agents Med Chem 8:351–357

    PubMed  CAS  Google Scholar 

  29. Strauss PR, Beard WA, Patterson TA, Wilson SH (1997) Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism. J Biol Chem 272:1302–1307

    Article  PubMed  CAS  Google Scholar 

  30. Adhikari S, Üren A, Roy R (2008) Dipole-dipole interaction stabilizes the transition state of apurinic/apyrimidinic endonuclease—abasic site interaction. J Biol Chem 283:1334–1339

    Article  PubMed  CAS  Google Scholar 

  31. Berquist BR, McNeill DR, Wilson DM 3rd (2008) Characterization of abasic endonuclease activity of human Ape1 on alternative substrates, as well as effects of ATP and sequence context on AP site incision. J Mol Biol 379:17–27

    Article  PubMed  CAS  Google Scholar 

  32. Wilson DM 3rd (2003) Properties of and substrate determinants for the exonuclease activity of human apurinic endonuclease Ape1. J Mol Biol 330:1027–1037

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Prof. Sankar Mitra of UTMB, Galveston, Texas for providing the mAPE1 cDNA. The authors thank Dr. Amrita Cheema for proteomics experiments performed at the Proteomics and Metabolomics Shared Resource of the Lombardi Comprehensive Cancer Center. The authors also thank Mrs. Jordan Woodrick for critically reading the paper. The study was supported by NIH grants RO1 CA 92306 (RR), RO1 CA 113447 (RR) and ACS grant ACS-IRG-92-152-17 (SA).

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Authors and Affiliations

  1. Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, LL level, S-122, 3800 Reservoir Road, Washington, DC, 20057, USA

    Sanjay Adhikari, Praveen Varma Manthena, Krishna Kiran Kota, Soumendra Krishna Karmahapatra, Gargi Roy, Rahul Saxena, Aykut Üren & Rabindra Roy

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  1. Sanjay Adhikari
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Correspondence to Rabindra Roy.

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Adhikari, S., Manthena, P.V., Kota, K.K. et al. A comparative study of recombinant mouse and human apurinic/apyrimidinic endonuclease. Mol Cell Biochem 362, 195–201 (2012). https://doi.org/10.1007/s11010-011-1142-5

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  • DOI: https://doi.org/10.1007/s11010-011-1142-5

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