1887

Abstract

The nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 3′–5′ exonucleases. The CfMNPV ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 3′–5′ exonuclease that cleaves oligonucleotides from the 3′ end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 5′-digoxigenin- or 5′-P-labelled oligo(dT) substrate was observed at increasing incubation times or increased amounts of V-TREX. The 3′-excision activity of V-TREX was maximal at alkaline pH (9·5) in the presence of 5 mM MgCl, 2 mM dithiothreitol and 0·1 mg BSA ml.

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2004-12-01
2019-11-17
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References

  1. Barnes, M. H., Spacciapoli, P., Li, D. H. & Brown, N. C. ( 1995; ). The 3′–5′ exonuclease site of DNA polymerase III from Gram-positive bacteria: definition of a novel motif structure. Gene 165, 45–50.[CrossRef]
    [Google Scholar]
  2. Bernad, A., Blanco, L., Lázaro, J. M., Martín, G. & Salas, M. ( 1989; ). A conserved 3′→5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59, 219–228.[CrossRef]
    [Google Scholar]
  3. 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.[CrossRef]
    [Google Scholar]
  4. Bronstein, J. C. & Weber, P. C. ( 1996; ). Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli. J Virol 70, 2008–2013.
    [Google Scholar]
  5. Bzymek, M., Saveson, C. J., Feschenko, V. V. & Lovett, S. T. ( 1999; ). Slipped misalignment mechanisms of deletion formation: in vivo susceptibility to nucleases. J Bacteriol 181, 477–482.
    [Google Scholar]
  6. Hoffmann, P. J. & Cheng, Y.-C. ( 1979; ). DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity. J Virol 32, 449–457.
    [Google Scholar]
  7. Kruchen, B. & Rueger, B. ( 2003; ). The DIG system – nonradioactive and highly sensitive detection of nucleic acids. Biochemica 3, 13–15.
    [Google Scholar]
  8. Lahue, R. S., Au, K. G. & Modrich, P. ( 1989; ). DNA mismatch correction in a defined system. Science 245, 160–164.[CrossRef]
    [Google Scholar]
  9. Lehman, I. R. & Nussbaum, A. L. ( 1964; ). The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase). J Biol Chem 239, 2628–2636.
    [Google Scholar]
  10. Li, L. & Rohrmann, G. F. ( 2000; ). Characterization of a baculovirus alkaline nuclease. J Virol 74, 6401–6407.[CrossRef]
    [Google Scholar]
  11. Mazur, D. J. & Perrino, F. W. ( 1999; ). Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3′→5′ exonucleases. J Biol Chem 274, 19655–19660.[CrossRef]
    [Google Scholar]
  12. Mazur, D. J. & Perrino, F. W. ( 2001; ). Excision of 3′ termini by the TREX1 and TREX2 3′→5′ exonucleases. Characterization of the recombinant proteins. J Biol Chem 276, 17022–17029.[CrossRef]
    [Google Scholar]
  13. Mikhailov, V. S., Okano, K. & Rohrmann, G. F. ( 2003; ). Baculovirus alkaline nuclease possesses a 5′→3′ exonuclease activity and associates with the DNA-binding protein LEF-3. J Virol 77, 2436–2444.[CrossRef]
    [Google Scholar]
  14. Mikhailov, V. S., Okano, K. & Rohrmann, G. F. ( 2004; ). Specificity of the endonuclease activity of the baculovirus alkaline nuclease for single-stranded DNA. J Biol Chem 279, 14734–14745.[CrossRef]
    [Google Scholar]
  15. Razavy, H., Szigety, S. K. & Rosenberg, S. M. ( 1996; ). Evidence for both 3′ and 5′ single-strand DNA ends in intermediates in chi-stimulated recombination in vivo. Genetics 142, 333–339.
    [Google Scholar]
  16. Scheuermann, R. H. & Echols, H. ( 1984; ). A separate editing exonuclease for DNA replication: the ε subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A 81, 7747–7751.[CrossRef]
    [Google Scholar]
  17. Slack, J. M., Ribeiro, B. M. & Lobo de Souza, M. ( 2004; ). The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus contains a 3′ repair exonuclease homologue and lacks v-cath and ChiA genes. J Gen Virol 85, 211–219.[CrossRef]
    [Google Scholar]
  18. Strauss, B. S., Sagher, D. & Acharya, S. ( 1997; ). Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA. Nucleic Acids Res 25, 806–813.[CrossRef]
    [Google Scholar]
  19. Taft-Benz, S. A. & Schaaper, R. M. ( 1998; ). Mutational analysis of the 3′-5′ proofreading exonuclease of Escherichia coli DNA polymerase III. Nucleic Acids Res 26, 4005–4011.[CrossRef]
    [Google Scholar]
  20. Viswanathan, M. & Lovett, S. T. ( 1999; ). Exonuclease X of Escherichia coli. A novel 3′→5′ DNase and DnaQ superfamily member involved in DNA repair. J Biol Chem 274, 30094–30100.[CrossRef]
    [Google Scholar]
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