BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and Characterization of a Deoxyriboendonuclease from Mycobacterium smegmatis

  • Received : 2005.08.27
  • Accepted : 2005.12.01
  • Published : 2006.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

A deoxyriboendonuclease has been purified to near homogeneity from a fast growing mycobacterium species, M. smegmatis and characterized to some extent. The size of enzyme is about 43 kDa as determined by a denaturing gel analysis. It shows optimum activity at $32^{\circ}C$ in Tris-HCl buffer (pH 7.2) containing 2.5 mM of $MgCl_2$. Both EDTA and $K^+$ but not $Na^+$ inhibit its activity. Evidences show that the enzyme is not a restriction endonuclease but catalyzes the endonucleolytic cleavage of both the double- as well as the single-strand DNA non-specifically. It has been shown that the cleavage by this enzyme generates DNA fragments carrying phosphate groups at 5' ends and hydroxyl group at the 3' ends, respectively. Analysis reveals that no endonuclease having size and property identical to our deoxyriboendonuclease had been purified from M. smegmatis before. The property of our enzymes closely matches with the deoxyriboendonucleases purified from diverse sources including bacteria.

Keywords

References

  1. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Brnakova, Z., Godany, A. and Timko, J. (2005) An extracellular endodeoxyribonuclease from Streptomyces aureofaciens. Biochim. Biophys. Acta 1721, 116-123 https://doi.org/10.1016/j.bbagen.2004.10.008
  3. Baranovskii, A. G., Buneva, V. N. and Nevinsky, G. A. (2004) Human deoxyribonucleases. Biochemistry 69, 587-601
  4. Chaudhuri, B., Sau, S., Datta, H. J. and Mandal, N. C. (1993) Isolation, characterization, and mapping of temperaturesensitive mutations in the genes essential for lysogenic and lytic growth of the mycobacteriophage L1. Virology 194, 166- 172 https://doi.org/10.1006/viro.1993.1246
  5. Chevalier, B. S. and Stoddard, B. L. (2001) Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 29, 3757- 3774 https://doi.org/10.1093/nar/29.18.3757
  6. Dryden, D. T., Murray, N. E. and Rao, D. N. (2001) Nucleoside triphosphate-dependent restriction enzymes. Nucleic Acids Res. 29, 3728-3741 https://doi.org/10.1093/nar/29.18.3728
  7. Ganesh, N. and Muniyappa, K. (2003) Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA. Proteins 53, 6-17 https://doi.org/10.1002/prot.10433
  8. Griffin, T. J. 4th, Parsons, L., Leschziner, A. E., DeVost, J., Derbyshire, K. M. and Grindley, N. D. (1999) In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis. Nucleic Acids Res. 27, 3859-3865 https://doi.org/10.1093/nar/27.19.3859
  9. Guhan, N. and Muniyappa, K. (2002) Mycobacterium tuberculosis RecA intein possesses a novel ATP-dependent site-specific double-stranded DNA endonuclease activity. J. Biol. Chem. 277, 16257-16264 https://doi.org/10.1074/jbc.M112365200
  10. Hatfull, G. F. (2000) Molecular genetics of mycobacteriophages; in Molecular Genetics of Mycobacteria, Hatfull, G. F. and Jacob, W. R., Jr. (eds.), pp 37- 54, ASM Press, Washington D. C., USA
  11. Kishi, K., Yasuda, T. and Takeshita, H. (2001) DNase I: structure, function, and use in medicine and forensic science. Leg Med. 3, 69-83 https://doi.org/10.1016/S1344-6223(01)00004-9
  12. Krogh, B. O. and Symington, L. S. (2004) Recombination proteins in yeast. Annu. Rev. Genet. 38, 233-271 https://doi.org/10.1146/annurev.genet.38.072902.091500
  13. Martinez-Canamero, M. M., Munoz, J., Extremera, A. L. and Arias, J. M. (1991) Deoxyribonuclease activities in Myxococcus coralloides D. J. Appl. Bacteriol. 71, 170-175 https://doi.org/10.1111/j.1365-2672.1991.tb02974.x
  14. Miller, E. S., Kutter, E., Mosig, G., Arisaka, F., Kunisawa, T. and Ruger, W. (2003) Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev. 67, 86-156 https://doi.org/10.1128/MMBR.67.1.86-156.2003
  15. Murmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 20-218
  16. Papavinasasundaram, K. G., Movahedzadeh, F., Keer, J. T., Stoker, N. G., Colston, M. J. and Davis, E. O. (1997). Mycobacterial recA is cotranscribed with a potential regulatory gene called recX. Mol. Microbiol. 24, 141-153 https://doi.org/10.1046/j.1365-2958.1997.3441697.x
  17. Pingoud, A. and Jeltsch, A. (2001) Structure and function of type II restriction endonucleases. Nucleic Acids Res. 29, 3705-3727 https://doi.org/10.1093/nar/29.18.3705
  18. Radding, C. M. (1964) Nuclease activity in defective lysogens of phage lambda. Biochem. Biophys. Res. Commun. 15, 8-12 https://doi.org/10.1016/0006-291X(64)90093-2
  19. Rama, J. M., Perez Urena, M. T., Lopez, P. and Espinosa, M. (1987) Characterization of intracellular deoxyribonucleases of Bacillus subtilis by SDS-polyacrylamide gel electrophoresis. Microbios. 49, 199-212
  20. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory Press, New York, USA
  21. Schofield, M. J. and Hsieh, P. (2003) DNA mismatch repair: molecular mechanisms and biological function. Annu. Rev. Microbiol. 57, 579-608 https://doi.org/10.1146/annurev.micro.57.030502.090847
  22. Shankar, S. and Tyagi, A. K. (1992) MhaAI, a novel isoschizomer of PstI from Mycobacterium habana recognizing 5'-CTGCA/G- 3'. Nucleic Acids Res. 20, 2891 https://doi.org/10.1093/nar/20.11.2891
  23. Shankar, S. and Tyagi, A. K. (1993a) Purification and characterization of restriction endonuclease MgoI from Mycobacterium gordonae. Gene 131, 153-154 https://doi.org/10.1016/0378-1119(93)90686-W
  24. Shankar, S. and Tyagi, A. K. (1993b) MchAI and MchAII, two class-II restriction endonucleases from Mycobacterium chelonei. Gene 132, 119-123. https://doi.org/10.1016/0378-1119(93)90523-6
  25. Starosciak, B. J. and Dobrzanski, W. T. (1980). Deoxyribonucleases of Streptococcus sanguis. Acta Microbiol. Pol. 29, 35-47
  26. Suri, R. (2005) The use of human deoxyribonuclease (rhDNase) in the management of cystic fibrosis. BioDrugs. 19, 135-144 https://doi.org/10.2165/00063030-200519030-00001
  27. Winder, F. G. and Lavin, M. F. (1971) Partial purification and properties of a nucleoside triphosphate-dependent deoxyribonuclease from Mycobacterium smegmatis. Biochim. Biophys. Acta. 247, 542-561 https://doi.org/10.1016/0005-2787(71)90691-5

Cited by

  1. Moderately thermostable phage Φ11 Cro repressor has novel DNA-binding capacity and physicochemical properties vol.42, pp.3, 2009, https://doi.org/10.5483/BMBRep.2009.42.3.160