The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Characterization of a Nuclease Gene of Chlorella Virus SS-2

  • Park, Yun-Jung (Department of Microbiology, Pukyong National University) ;
  • Jung, Sang-Eun (Department of Microbiology, Pukyong National University) ;
  • Choi, Tae-Jin (Department of Microbiology, Pukyong National University)
  • Published : 2009.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

Keywords

References

  1. Agarkova, I. V., Dunigan, D. D. and Van Etten, J. L. 2006. Virionassociated restriction endonucleases of chloroviruses. J Viral. 80:8114-8123 https://doi.org/10.1128/JVI.00486-06
  2. Anderson, J. E. 1993. Restriction endonucleases and modification methy1ases. Curr. Opin. Struct. BioI. 3:24-30 https://doi.org/10.1016/0959-440X(93)90197-S
  3. Bujnicki, J. M. and Rychlewski, L. 2001. The herpesvirus alkaline exonuclease belongs to the restriction endonuclease PO-(DIE)XK superfamily: insight from molecular modeling and phylogenetic analysis. Virus Genes 22:219-230 https://doi.org/10.1023/A:1008131810233
  4. Burbank, D. E., Shields, S. L., Schuster, A. M. and Van Etten, J. L. 1990. 5-Azacytidine resistant mutants of ChI orella virus IL-3A. Virology 176:311-315 https://doi.org/10.1016/0042-6822(90)90261-O
  5. Carter, D. and Radding, C. 1971. The role of exonuclease and beta protein of phage lambda in genetic recombination. II. substrate spcciticity and the mode of action of lambda exonuclease. J. Biol. Chem. 246:2502-2512
  6. Cheng. Y-C., Chen, J.-Y, Hoffinann, P. J. and Glaser, R. 1980. Studies on the activity of DNase associated with the replication of the Epstein-Barr virus. Virology 100:334-338 https://doi.org/10.1016/0042-6822(80)90524-3
  7. Cho, H.-H., Park, H.-H., Kim, J.-O. and Choi, T.-J. 2002. Isolation and characterization of Chlorella viruses from freshwater sources in Korea. Mol. Cells 14:168-176
  8. Doetsch, P. W. and Cunningham, R. P. 1990. The enzymology of apurinic/apyrimidinic endonucleases. Mutat. Res. 236: 173-201 https://doi.org/10.1016/0921-8777(90)90004-O
  9. Dunigan, D. D., Fitzgerald, L. A. and Van Etten, J. L. 2006. Phycodnaviruses: a peek at genetic diversity. Virus Res. 117: 119-132 https://doi.org/10.1016/j.virusres.2006.01.024
  10. Fitzgerald, L. A .. Graves, M. V., Li, X., Feldblyum, T., Hartigan, J. and Van Etten, J. L. 2007. Sequence and annotation of the 3l4-kb MT325 and the 321-kb FR483 viruses that infect Chlorella Pbi. Virology 358:459-471 https://doi.org/10.1016/j.virol.2006.08.034
  11. Kang, M., Dunigan, D. D. and Van Etten, J. L. 2005. Chlorovirus: a genus of phycodnaviridae that infects certain chlorella-like green algae. Mol. Plant Pathol. 6:213-224 https://doi.org/10.1111/j.1364-3703.2005.00281.x
  12. Knizewski, L. Kinch, L. Grishin, N. Rychlewski, L. and Ginalski, K. 2006. Human hcrpcsvirus I UL24 gcne encodes a potential PD-(D/E)XK endonuclease. J. Virol. 80:2575-2577 https://doi.org/10.1128/JVI.80.5.2575-2577.2006
  13. Kovall, R. A. and Matthews, B. W. 1999. Type II restriction endonucleases: structural, functional and evolutionary relationships. Curr. Opin. Chem. BioI. 3:578-583 https://doi.org/10.1016/S1367-5931(99)00012-5
  14. Landstein, D., Burband, D. E., Nietfeldt, J. W. and Van Etten, J. L. 1995. Large deletions in antigenic variants of the Chiarella vims PBeY-I. Virology 214:413-420 https://doi.org/10.1006/viro.1995.0051
  15. Li, L. and Rohrmann, G. F. 2000. Characterization of a baculovirus alkaline nucleasc. J. Viral. 74:6401-6407 https://doi.org/10.1128/JVI.74.14.6401-6407.2000
  16. Li, Y, Lu, Z., Sun, L., Ropp, S., Kutish, G. E, Rock, D. L. and Van Etten, J. L. 1997. Analysis of 74 kb of DNA located at the right end ofthe 330-kb chlorella virus PBCV-I genome. Virology 237:360-377 https://doi.org/10.1006/viro.1997.8805
  17. Liu, M.-T., Hu, H.-P., Hsu, T.-Y and Chen, J.-Y 2003. Sitedirected mutagenesis in a conserved motif of Epstein-Barr virus DNase that is homologous to the catalytic centre of type II restriction cndonucleascs . .J Gen. Virol. 84:677-686 https://doi.org/10.1099/vir.0.18739-0
  18. Murzin, A. G, Brenner, S. E., Hubbard, T. and Chothia, C. 1995. SCOP: a stmctural classification of proteins database for the investigation of sequences and structures. J. Mol. Bioi. 247:536-540
  19. Nishida, K., Kimura, Y, Kawasaki, T., Fujie, M. and Yamada, T. 1999. Genetic variation of chlorella viruses: variable regions localized on the CVK2 genomic DNA. Virology 255:376-384 https://doi.org/10.1006/viro.1998.9595
  20. Plchova, H., Hartung, E and Puchta, H. 2003. Biochemical characterization of an exonuclease from Arabidopsis tha/tana reveals similarities to the DNA exonuclease of the human werner syndrome protein. J. BioI. Chem. 278:44128-44138 https://doi.org/10.1074/jbc.M303891200
  21. Ponchon, L., Boulanger, P., Labesse, G. and Letellier, L. 2006. The endonuclease domain of bacteriophage termmases belong to the resolvase/integrase/ribonuclease H superfamily. J Bioi. Chem. 281 :5829-5836 https://doi.org/10.1074/jbc.M511817200
  22. Sam brook, J. and Russell, D. W. 2001. Molecular cloning; A laboratory Manual, 3ed ed., CSH Laboratory Press, Cold Spring Harbor, NY
  23. Selent, U., Ruter, T., Kohler, E., Liedtke, M., Thielking, Y, Alves, J. Oelgeschlager, T. Wolfes, H., Peters, F. and Pingoud, A. 1992. A site-directed mutagenesis study to identifY amino acid residues involved in the catalytic function of the restriction endonuclease EcoR Y. Biochemistry 31 :4808-4815 https://doi.org/10.1021/bi00135a010
  24. Shen, J.-C. and Loeb, L. A. 2000. Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA. Nucleic Acids Res. 28:3260-3268 https://doi.org/10.1093/nar/28.17.3260
  25. Van Etten, J. L. and Meints, R. H. 1999. Giant viruses infecting algae. Annu. Rev. Microbial. 53:447-494 https://doi.org/10.1146/annurev.micro.53.1.447
  26. Van Etten, J. L., Burbank, D. E., Xia, Y and Meints, R. H. 1983. Growth cycle of a virus, PBCV-I, that infects chlorella-like algae. Virology 126:117-125 https://doi.org/10.1016/0042-6822(83)90466-X
  27. Weller, S. K., Seghatoleslami, R. M., Shao, L., Rowse, D. and Carmichael. E. P. 1990. The herpes simplex virus type I alkaline nuclease is not essential for viral DNA synthesis: isolation and characterization of a lacZ insertion mutant. J. Gen. Viral. 71 :2941-2952 https://doi.org/10.1099/0022-1317-71-12-2941
  28. Wolfes, B., Alves, J., Fliess, A., Geiger, R. and Pingoud, A. 1986. Site-directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential far endonucleolytic activity of EcoR I. Nucleic Acids Res. 14:9063-9080 https://doi.org/10.1093/nar/14.22.9063
  29. Xia, Y. and Van Etten, J. L. 1986. DNA methyltransferase induced by PBCY-I virus infection of a chlorella-Iike green alga. Mol. Cell. Biol. 6:1440-1445 https://doi.org/10.1128/MCB.6.5.1440
  30. Xia, Y, Burbank, D. E., Uher, L., Rabussay, D. and Van Etten, J. L. 1986. Restriction endonuclease activity induced by PBCVJ virus in fection of a chI orella-like green alga. Mol. Cell. Bioi. 6: 1430-1439 https://doi.org/10.1128/MCB.6.5.1430
  31. Zhang, Y., Nelson, M., Nietfeldt, J., Xia, Y, Burbank, D. E., Ropp, S. and Van Etten, J. L. 1998. Chi orella virus NY-2A encodes at least twelve DNA endonuclease/methyltransferase genes. Virology 240:366-375 https://doi.org/10.1006/viro.1997.8936
  32. Zhang, Y, Strasser, P., Grabherr, R. and Van Etten, J. L. 1994. Hairpin loop structure at the termini of the chlorella virus PBCV-l genome. Virology 202: 1079-1082 https://doi.org/10.1006/viro.1994.1444