Chinese Science Bulletin

, Volume 50, Issue 4, pp 311–316 | Cite as

Construction of DNA damage response genepprI function-deficient and function-complementary mutants inDeinococcus radiodurans

  • Guanjun Gao
  • Huiming Lu
  • Lifen Huang
  • Yuejin HuaEmail author


PprI, a DNA damage response factor from the extraordinary radioresistant bacteriumDeinococcus radi-odurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with theD. radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into thepprI locus in the genome of the wild-type strain R1. The resultingpprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the recombinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance inD. radiodurans. The fragments containing completepprI gene and 3′-terminal deletionpprI † were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI† were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-typeD. raiodurans Rl, whereas YR1 pRADKpprI† failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants inD. radiodurans is not only useful to elucidating the relationship between domains and functions of Pprl protein, but also opens the door to the further studies of the biological functions of PprI proteinin vivo.


Deinococcus radiodurans Rl pprI mutants complementary 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Battista, J. R., Earl, A. M., Park, M. J., Why isDeinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol., 1999, 7: 362–365.PubMedCrossRefGoogle Scholar
  2. 2.
    Minton, K. W., DNA repair in the extremely radioresistant bacteriumDeinococcus radiodurans, Mol. Microbiol., 1994, 13: 9–15.PubMedCrossRefGoogle Scholar
  3. 3.
    Anderson, A. W., Nordan, H. C., Cain, R. F. et al., Studies on the radio-resistant micrococcus. I. The isolation, morphology, cultural characteristics and resistance to g radiation, Food Technol., 1956, 10: 575–578.Google Scholar
  4. 4.
    Makarova, K. S., Aravind, L., Wolf, Y. I. et al., Genome of the extremely radiation-resistant bacteriumDeinococcus radiodurans viewed from the perspective of comparative genomics, Microbiol. Mol. Biol. Rev, 2001, 65: 44–79.PubMedCrossRefGoogle Scholar
  5. 5.
    White, O., Eisen, J. A., Heidelberg, J. F. et al., Genome sequence of the radio-resistant bacteriumDeinococcus radiodurans R1, Science, 1999, 286: 1571–1577.PubMedCrossRefGoogle Scholar
  6. 6.
    Liu, Y. Q., Zhou, J. Z., Omelchenko, M. V. et al., Transcriptome dynamics ofDeinococcus radiodurans recovering from ionizing radiation, Proc. Natl. Acad. Sci. USA, 2003, 100: 4191–4196.PubMedCrossRefGoogle Scholar
  7. 7.
    Levin-Zaidman, S., Englander, J., Shimoni, E. et al., Ringlike structure of theDeinococcus radiodurans genome: A key to radioresistance? Science, 2003, 299: 254–256.PubMedCrossRefGoogle Scholar
  8. 8.
    Battista, J. R., Cox, M. M., Daly, M. J. et al., The structure ofD. radiodurans, Science, 2004, 302: 567–568.CrossRefGoogle Scholar
  9. 9.
    Daly, M. J., Gaidamakova, E. K., Matrosova, V. Y. et al., Accumulation of Mn(II) inDeinococcus radiodurans facilitates gamma-radiation resistance, Science, 2004, 306: 1025–1028.PubMedCrossRefGoogle Scholar
  10. 10.
    Kim, J., Cox, M. M., The RecA protein ofDeinococcus radiodurans andEscherichia coli promote DNA strand exchange via inverse pathways, Proc. Natl. Acad. Sci. USA, 2002, 99: 7917–7921.PubMedCrossRefGoogle Scholar
  11. 11.
    Satoh, K., Narumi, I., Kikuchi, M. et al., Characterization of RecA424 and RecA670 proteins fromDeinococcus radiodurans, J. Biochem., 2002, 131: 121–129.PubMedGoogle Scholar
  12. 12.
    Hua, Y. J., Narumi, I., Gao, G. J. et al., PprI: A general switch responsible extreme radioresistance ofDeinococcus radiodurans, Biochem. Biophy. Res. Commun., 2003, 306: 354–360.CrossRefGoogle Scholar
  13. 13.
    Gao, G. J., Tian, B., Liu, L. L., et al., Expression ofDeinococcus radiodurans PprI enhances the radioresistance ofEscherichia coli, DNA Repair, 2003, 2: 1419–1427.PubMedCrossRefGoogle Scholar
  14. 14.
    Earl, A. M., Mohundro, M. M., Mian, I. S. et al., The IrrE protein ofDeinococcus radiodurans Rl is a novel regulator of recA expression, J. Bacteriol., 2002, 184: 6216–6224.PubMedCrossRefGoogle Scholar
  15. 15.
    Hua, Y. J., Gao, G. J., Comparative genomics of genes contributing to DNA repair the in radiation-resistantDeinococcus radiodurans, Acta Microbiol. Sinica, 2003, 43: 120–126.Google Scholar
  16. 16.
    Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning, A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1982.Google Scholar
  17. 17.
    Meima, R., Rothfuss, H. M., Gewin, L. et al., Promoter cloning in the radioresistant bacteriumDeinococcus radiodurans. J. Bacteriol., 2001, 183: 3169–3175.PubMedCrossRefGoogle Scholar
  18. 18.
    Wood, R. D., Mitchell, M., Sgouros, J. et al., Human DNA repair genes, Science, 2001, 291: 1284–1289.PubMedCrossRefGoogle Scholar
  19. 19.
    Brim, H., McFarlan, S. C., Fredrickson, J. K. et al., EngineeringDeinococcus radiodurans for metal remediation in radioactive mixed waste environments, Nat. Biotechnol., 2000, 18: 85–90.PubMedCrossRefGoogle Scholar
  20. 20.
    Lange, C. C., Wackett, L. P., Minton, K. W. et al., Engineering a recombinantDeinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments, Nat. Biotechnol., 1998, 16: 929–933.PubMedCrossRefGoogle Scholar

Copyright information

© Science in China Press 2005

Authors and Affiliations

  • Guanjun Gao
    • 1
  • Huiming Lu
    • 1
  • Lifen Huang
    • 1
  • Yuejin Hua
    • 1
    Email author
  1. 1.Institute of Nuclear-Agricultural ScienceZhejiang UniversityHangzhouChina

Personalised recommendations