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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Biochemical Study of Recombinant PcrA from Staphylococcus aureus for the Development of Screening Assays

  • Dubaele, Sandy (Novartis Institutes for BioMedical Research, Oncology Research) ;
  • Martin, Christophe (Novartis Institutes for BioMedical Research, Oncology Research) ;
  • Bohn, Jacqueline (Novartis Institutes for BioMedical Research, Oncology Research) ;
  • Chene, Patrick (Novartis Institutes for BioMedical Research, Oncology Research)
  • Published : 2007.01.31
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Abstract

Helicases are ubiquitous enzymes, which utilize the energy liberated during nucleotide triphosphate hydrolysis to separate double-stranded nucleic acids into single strands. These enzymes are very attractive targets for the development of new antibacterial compounds. The PcrA DNA helicase from Staphylococcus aureus is a good candidate for drug discovery. This enzyme is unique in the genome of S. aureus and essential for this bacterium. Furthermore, it has recently been published that it is possible to identify inhibitors of DNA helicases such as PcrA. In this report, we study the properties of recombinant PcrA from S. aureus purified from Escherichia coli to develop ATPase and helicase assays to screen for inhibitors.

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References

  1. Anand, S. P. and Khan, S. A. (2004) Structure-specific DNA binding and bipolar helicase activities of PcrA. Nucleic Acids Res. 32, 3190-3197. https://doi.org/10.1093/nar/gnh012
  2. Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K., Oguchi, A., Nagai, Y., Iwama, N., Asano, K., Naimi, T., Kuroda, H., Cui, L., Yamamoto, K. and Hiramatsu, K. (2002) Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359, 1819-1827. https://doi.org/10.1016/S0140-6736(02)08713-5
  3. Bird, L. E., Brannigan, J. A., Subramanya, S. H. and Wigley, D. B. (1998) Characterisation of Bacillus stearothermophillus PcrA helicase: evidence against an active rolling mechanism. Nucleic Acid Res. 26, 2686-2693. https://doi.org/10.1093/nar/26.11.2686
  4. Chang, S., Sievert, D. M., Hageman, J. C., Boulton, M. L., Tenover, F. C., Downes, F. P., Shah, S., Rudrik, J. T., Pupp, G. R., Brown, W. J., Cardo, D. and Fridkin, S. K. (2003) Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N. Engl. J. Med. 348, 1342-1347. https://doi.org/10.1056/NEJMoa025025
  5. Chang, T. L., Naqvi, A., Anand, S. P., Kramer, M. G., Munshi, R. and Khan, S. A. (2002) Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication. J. Biol. Chem. 277, 45880-45886. https://doi.org/10.1074/jbc.M207383200
  6. Cooke, J. (2004) Infectious diseases - the need for new antibiotics. Hospital Pharmacist 11, 265-268.
  7. Dillingham, M. S., Soultanas, P. and Wigley, D. B. (1999) Sitedirected mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation. Nucleic Acids Res. 27, 3310-3317. https://doi.org/10.1093/nar/27.16.3310
  8. Dubaele, S., Jahnke, W., Schoepfer, J., Fuchs, J. and Chene, P. (2006) Inhibition of DNA helicases with DNA-competitive inhibitors. Bioorg. Med. Chem. Lett. 16, 923-927. https://doi.org/10.1016/j.bmcl.2005.10.110
  9. Garcia-Lara, J., Masalha, M. and Foster, S. J. (2005) Staphylococcus aureus: the search for novel targets. Drug Disc Today 10, 643-651. https://doi.org/10.1016/S1359-6446(05)03432-X
  10. Gill, S. R., Fouts, D. E., Archer, G. L., Mongodin, E. F., Deboy, R. T., Ravel, J., Paulsen, I. T., Kolonay, J. F., Brinkac, L., Beanan, M., Dodson, R. J., Daugherty, S. C., Madupu, R., Angiuoli, S. V., Durkin, A. S., Haft, D. H., Vamathevan, J., Khouri, H., Utterback, T., Lee, C., Dimitrov, G., Jiang, L., Qin, H., Weidman, J., Tran, K., Kang, K., Hance, I. R., Nelson, K. E. and Fraser, C. M. (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilmproducing methicillin-resistant Staphylococcus epidermidis strain. J. Bacteriol. 187, 2426-2438. https://doi.org/10.1128/JB.187.7.2426-2438.2005
  11. Holden, M. T. G., Feil, E. J., Lindsay, J. A., Peacock, S. J., Day, N. P. J., Enright, M. C., Foster, T. J., Moore, C. E., Hurst, L., Atkin, R., Barron, A., Bason, N., Bentley, S. D., Chillingworth, C., Chillingworth, T., Churcher, C., Clark, L., Corton, C., Cronin, A., Doggett, J., Dowd, L., Feltwell, T., Hance, Z., Harris, B., Hauser, H., Holroyd, S., Jagels, K., James, K. D., Lennard, N., Line, A., Mayes, R., Moule, S., Mungall, K., Ormond, D., Quail, M. A., Rabbinowitsch, E., Rutherford, K., Sanders, M., Sharp, S., Simmonds, M., Stevens, K., Whitehead, S., Barrell, B. G., Spratt, B. G. and Parkhill, J. (2004) Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the rapid evolution of virulence and drug resistance. Proc. Natl. Acad. Sci. USA 101, 9786-9791. https://doi.org/10.1073/pnas.0402521101
  12. Iordanescu, S. (1993a) Characterization of the Staphylococcus aureus chromosomal gene PcrA, identified by mutations affecting plasmid pT181 replication. Mol. Gen. Genet. 241, 185-192. https://doi.org/10.1007/BF00280216
  13. Iordanescu, S. (1993b) Plasmid pT181-linked suppressors of Staphylococcus aureus PcrA3 chromosomal mutation. J. Bacteriol. 175, 3916-3917. https://doi.org/10.1128/jb.175.12.3916-3917.1993
  14. Ji, Y., Zhang, B., Van Horn, S. F., Warren, P., Woodnutt, G., Burnham, M. K. R. and Rosenberg, M. (2001) Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293, 2266-2269. https://doi.org/10.1126/science.1063566
  15. Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004) Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase and phosphatase. Anal. Chem. 76, 1429-1436. https://doi.org/10.1021/ac030234b
  16. Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I., Cui, L., Oguchi, A., Aoki, K., Nagai, Y., Lian, J., Ito, T., Kanamori, M., Matsumaru, H., Maruyama, A., Murakami, H., Hosoyama, A., Mizutani-Ui, Y., Takahashi, N. K., Sawano, T., Inoue, R., Kaito, C., Sekimizu, K., Hirakawa, H., Kuhara, S., Goto, S., Yabuzaki, J., Kanehisa, M., Yamashita, A., Oshima, K., Furuya, K., Yoshino, C., Shiba, T., Hattori, M., Ogasawara, N., Hayashi, H. and Hiramatsu, K. (2001) Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357, 1225-1240.礙돀샊?⨀堘?⨀想?⨀儙돐?⨀?⨀ꃳ?⨀⤙댐＃Ȁ https://doi.org/10.1016/S0140-6736(00)04403-2
  17. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. and Candia, O. A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95-97. https://doi.org/10.1016/0003-2697(79)90115-5
  18. Le Loir, Y., Baron, F. and Gautier, M. (2003) Staphylococcus aureus and food poisoning. Genet. Mol. Res. 2, 63-76.
  19. Livermore, D. M. (2004) The need for new antibiotics. Clin. Microbiol. Infect. 10, 1-9.
  20. Naqvi, A., Tinsley, E. and Khan, S. A. (2003) Purification and characterization of the PcrA helicase of Bacillus anthracis. J. Bacteriol. 185, 6633-6639. https://doi.org/10.1128/JB.185.22.6633-6639.2003
  21. Notredame, C., Higgins, D. and Heringa, J. (2000) T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217. https://doi.org/10.1006/jmbi.2000.4042
  22. Petit, M. A. and Ehrlich, D. (2002) Essential bacterial helicases that counteract the toxicity of recombination proteins. EMBO J. 21, 3137-3147. https://doi.org/10.1093/emboj/cdf317
  23. Soultanas, P., Dillingham, M. S., Velankar, S. S. and Wigley, D. B. (1999) DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase. J. Mol. Biol. 290, 137-148. https://doi.org/10.1006/jmbi.1999.2873
  24. Todd, J. K. (2005) Staphylococcal infections. Pediatr. Rev. 26, 444-450.
  25. Wardell, A. D., Errington, W., Ciaramella, G., Merson, J. and McGarvey, M. J. (1999) Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein. J. Gen. Virol. 80, 701-709. https://doi.org/10.1099/0022-1317-80-3-701

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