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Molecular Biology Reports

, Volume 10, Issue 2, pp 91–97 | Cite as

DNA-binding proteins of human placenta: purification and characterization of an endonuclease

  • T. Premeela
  • A. R. A. Rajakumar
  • G. Shanmugam
Article

Abstract

DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125–0.15M potassium phosphate. PC fractions containing electrophoretically pure 34KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3–5 mM Mg2+ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate.

Keywords

Potassium Phosphate Ammonium Sulphate Tetraacetate Ethylenediamine Tetraacetate Ammonium Sulphate Fractionation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    GeiderK. & Hoffmann-BerlingH., 1981. Ann. Rev. Biochem. 50: 253–260.Google Scholar
  2. 2.
    FalaschiA., CobianchiF. & RivaS., 1980. Trends Biochem. Sci. 5: 154–157.Google Scholar
  3. 3.
    TsaiR. I. & GreenH., 1973. J. Mol. Biol. 73: 307–316.Google Scholar
  4. 4.
    McEnteeK., WeinstockG. M. & LehmanI. R., 1981. J. Biol. Chem. 256: 8835–8844.Google Scholar
  5. 5.
    AraiK., YasudaS. & KornbergA., 1981. J. Biol. Chem. 256: 5247–5252.Google Scholar
  6. 6.
    SigalN., DeliusH., KornbergT., GefterM. L., AlbertsB. M., 1972. Proc. Natl. Acad. Sci. U.S.A. 69: 3537–3541.Google Scholar
  7. 7.
    SuginoA. & CozzarelliN. R., 1980. J. Biol. Chem. 255: 6299–6306.Google Scholar
  8. 8.
    HerrickG. & AlbertsB. M., 1976. J. Biol. Chem. 251: 2124–2132.Google Scholar
  9. 9.
    RajakumarA. R. A. & ShanmugamG., 1983. Exp. Cell. Res. 147: 119–125.Google Scholar
  10. 10.
    SheltonI. H., KasupskiG. J.Jr., OblinC. & HondR., 1981. Canad. J. Biochem. 59: 122–130.Google Scholar
  11. 11.
    GidoniD., SchellerA., BarnetB., HantzopolousP., OrenM. & PrivesC., 1982. J. Virol. 42: 456–466.Google Scholar
  12. 12.
    ShanmugamG., BhaduriS., ArensM. & GreenM., 1975. Biochemistry 14: 322–337.Google Scholar
  13. 13.
    MoranelliF. & LiebermanM. N., 1980. Proc. Natl. Acad. Sci. U.S.A. 77: 3201–3205.Google Scholar
  14. 14.
    ShaperN. L., GrafstormR. H. & GrossmanL., 1982. J. Biol. Chem. 257: 13455–13458.Google Scholar
  15. 15.
    LinsleyN. S., PenhoetE. E. & LinnS., 1977. J. Biol. Chem. 252: 1235–1242.Google Scholar
  16. 16.
    FeldbergR. S., LucasJ. L. & DannenbergA., 1982. J. Biol. Chem. 252: 6394–6401.Google Scholar
  17. 17.
    BoimeI., SzczesnaE. & SmithD., 1977. Eur. J. Biochem. 73: 515–520.Google Scholar
  18. 18.
    SzczesnaE. & BoimeI., 1976. Proc. Natl. Acad. Sci. U.S.A. 73: 1179–1183.Google Scholar
  19. 19.
    BlackburnP., WilsonG. & MooreS., 1977. J. Biol. Chem. 252: 5904–5910.Google Scholar
  20. 20.
    JacksonV. & ChalkleyR. 1981. J. Biol. Chem. 256: 5095–5103.Google Scholar
  21. 21.
    LowryO. H., RosenbergN. J., FarA. L. & RandallR. J., 1951. J. Biol. Chem. 193: 265–275.Google Scholar
  22. 22.
    PeacockA. C. & DingmanC. W., 1968. Biochemistry 7: 668–682.Google Scholar
  23. 23.
    LaemmliU. K. & FavreM., 1973. J. Mol. Biol. 80: 575–599.Google Scholar
  24. 24.
    DuguetM., BonneC. & deRecondoA., 1981. Biochemistry, 20: 3598–3603.Google Scholar
  25. 25.
    BanksG. R. & SpanosA., 1975. J. Mol. Biol. 93: 63–77.Google Scholar
  26. 26.
    PlankS. R. & WilsonS. H., 1980. J. Biol. Chem. 255: 11547–11556.Google Scholar
  27. 27.
    BeardP., MarrowJ. F. & BergP., 1973. J. Virol. 12: 1303–1313.Google Scholar
  28. 28.
    MachrayG. C. & BonnerJ., 1981. Biochemistry 20: 5466–5470.Google Scholar
  29. 29.
    WangE. C. & FurthJ. J., 1977. J. Biol. Chem. 252: 116–124.Google Scholar
  30. 30.
    LavinM. F., KikuchiT., CouncilmanC., JenkinsA., WinzerD. J. & KidsonC., 1976. Biochemistry 15: 2409–2414.Google Scholar
  31. 31.
    CordisG. A., GoldblattP. J. & DeutscherM. P., 1975. Biochemistry 14: 2596–2603.Google Scholar
  32. 32.
    KueblerJ. P. & GoldwaitD. A. 1977. Biochemistry 16: 1370–1377.Google Scholar
  33. 33.
    BlairD. B., ClewellD. B., SherrattD. J. & HelinskiD. R., 1971. Proc. Natl. Acad. Sci. U.S.A. 68: 210–214.Google Scholar
  34. 34.
    CleaverJ. E., 1974. Adv. Radiat. Biol. 5: 1–75.Google Scholar

Copyright information

© Dr W. Junk Publishers 1984

Authors and Affiliations

  • T. Premeela
    • 1
  • A. R. A. Rajakumar
    • 1
  • G. Shanmugam
    • 1
  1. 1.Cancer Biology Unit, School of Biological SciencesMadurai Kamaraj UniversityMaduraiIndia

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