Detection and Quantitation of RecBCD Enzyme (Exonuclease V) Activity

  • Douglas A. Julin
Part of the Methods in Molecular Biology™ book series (MIMB, volume 152)


The RecBCD enzyme serves two functions in the bacterial cell: it is a nuclease that destroys linear double-stranded DNA (dsDNA), and a DNA helicase that generates single-stranded DNA (ssDNA) used by RecA protein to initiate homologous recombination (1, 2, 3). A specific DNA sequence called Chi (5′-GCTGGTGG) is a signal that regulates these two functions (1,3). An encounter with Chi by RecBCD during its reaction with dsDNA leads to suppression of the nuclease activity and reveals the recombination-initiating function of RecBCD (1,3). The enzyme in Escherichia coli and other bacteria consists of three protein subunits encoded by the recB, recC, and recD genes, whereas some bacteria (e.g., Bacillus subtilis, Lactococcus lactis) produce a two-subunit enzyme [AddAB and RexAB, respectively (4,5)]. The genes encoding these latter enzymes complement an E. coli recBCD deletion mutation in vivo (5,6), and the purified AddAB enzyme has similar catalytic activity to the more extensively studied E. coli enzyme (7).


Nuclease Activity Circular ssDNA Linear dsDNA Circular dsDNA Nuclease Cleavage 
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  1. 1.
    Myers, R. S. and Stahl, F. W. (1994) k and the RecBC D enzyme of Escherichia coli. Annu. Rev. Genet. 28, 49–70.PubMedCrossRefGoogle Scholar
  2. 2.
    Smith, G. R., Amundsen, S. K., Dabert, P., and Taylor, A. F. (1995) The initiation and control of homologous recombination in Escherichia coli. Phil. Trans. R. Soc. Lond. B. 347, 13–20.CrossRefGoogle Scholar
  3. 3.
    Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., et al. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol. Rev. 58, 401–465.PubMedGoogle Scholar
  4. 4.
    Kooistra, J. and Venema, G. (1991) Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis. J. Bacteriol. 173, 3644–3655.PubMedGoogle Scholar
  5. 5.
    El Karoui, M., Ehrlich, D., and Gruss, A. (1998) Identification of the lactococcal exonuclease/recombinase and its modulation by the putative Chi sequence. Proc. Natl. Acad. Sci. USA 95, 626–631.PubMedCrossRefGoogle Scholar
  6. 6.
    Kooistra, J., Haijema, B. J., and Venema, G. (1993) The Bacillus subtilis addAB genes are fully functional in Escherichia coli. Mol. Microbiol. 7, 915–923.PubMedCrossRefGoogle Scholar
  7. 7.
    Shemyakin, M. F., Grepachevsky, A. A., and Chestukhin, A. V. (1979) Properties of Bacillus subtilis ATP-dependent deoxyribonuclease. Eur. J. Biochem. 98, 417–423.PubMedCrossRefGoogle Scholar
  8. 8.
    Smith, G. R. (1990) RecBCD Enzyme, in Nucleic Acids and Molecular Biology, vol. 4 (Eckstein, F. and Lilley, D. M. J., eds.), Springer-Verlag, Berlin, Heidelberg, pp. 78–98.Google Scholar
  9. 9.
    Muskavitch, K. M. T. and Linn, S. (1981) recBC-like enzymes: Exonuclease V deoxyribonucleases, in The Enzymes, vol. 14 (Boyer, P.D., ed.), Academic Press, New York, pp. 233–250.Google Scholar
  10. 10.
    Ponticelli, A. S., Schultz, D. W., Taylor, A. F., and Smith, G. R. (1985) Chidependent DNA strand cleavage by RecBC enzyme. Cell 41, 145–151.PubMedCrossRefGoogle Scholar
  11. 11.
    Dixon, D. A. and Kowalczykowski, S. C. (1993) The recombination hotspot ÷ is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell 73, 87–96.PubMedCrossRefGoogle Scholar
  12. 12.
    Oishi, M. (1969) An ATP-dependent deoxyribonuclease from Escherichia coli with a possible role in genetic recombination. Proc. Natl. Acad. Sci. USA 64, 1292–1299.PubMedCrossRefGoogle Scholar
  13. 13.
    Barbour, S. D. and Clark, A. J. (1970) Biochemical and genetic studies of recombination proficiency in Escherichia coli, I. Enzymatic activity associated with recB+ and recC+ genes. Proc. Natl. Acad. Sci. USA 65, 955–961.PubMedCrossRefGoogle Scholar
  14. 14.
    Wright, M., Buttin, G., and Hurwitz, J. (1971) The isolation and characterization from Escherichia coli of an adenosine triphosphate-dependent deoxyribonuclease directed by rec B, C genes. J. Biol. Chem. 246, 6543–6555.PubMedGoogle Scholar
  15. 15.
    Goldmark, P. J. and Linn, S. (1972) Purification and properties of the recBC DNase of Escherichia coli K-12. J. Biol. Chem. 247, 1849–1860.PubMedGoogle Scholar
  16. 16.
    Chen, H.-W., Randle, D. E., Gabbidon, M., and Julin, D. A. (1998) Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli. J. Mol. Biol. 278, 89–104.PubMedCrossRefGoogle Scholar
  17. 17.
    Rosamond, J., Telander, K. M., and Linn, S. (1979) Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+. J. Biol. Chem. 254, 8646–8652.PubMedGoogle Scholar
  18. 18.
    MacKay, V. and Linn, S. (1974) The mechanism of degradation of duplex deoxyribonucleic acid by the recBC enzyme of Escherichia coli K-12. J. Biol. Chem. 249, 4286–4294.PubMedGoogle Scholar
  19. 19.
    Taylor, A. and Smith, G. R. (1980) Unwinding and rewinding of DNA by the RecBC enzyme. Cell 22, 447–457.PubMedCrossRefGoogle Scholar
  20. 20.
    Taylor, A. F. and Smith, G. R. (1985) Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli. J. Mol. Biol. 185, 431–443.PubMedCrossRefGoogle Scholar
  21. 21.
    Lohman, T. M. and Bjornson, K. P. (1996) Mechanism of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214.PubMedCrossRefGoogle Scholar
  22. 22.
    Eggleston, A. K. and Kowalczykowski, S. C. (1993) Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks k-specific, but not non-specific, nuclease activity. J. Mol. Biol. 231, 605–620.PubMedCrossRefGoogle Scholar
  23. 23.
    Muskavitch, K. M. T. and Linn, S. (1982)A unified mechanism for the nuclease and unwinding activities of the recBC enzyme of Escherichia coli. J. Biol. Chem. 257, 2641–2648.PubMedGoogle Scholar
  24. 24.
    MacKay, V. and Linn, S. (1976) Selective inhibition of the DNase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. J. Biol. Chem. 251, 3716–3719.PubMedGoogle Scholar
  25. 25.
    Korangy, F. and Julin, D. A. (1994) Efficiency of ATP Hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry 33, 9552–9560.PubMedCrossRefGoogle Scholar
  26. 26.
    Roman, L. J. and Kowalczykowski, S. C. (1989) Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry 28, 2863–2873.PubMedCrossRefGoogle Scholar
  27. 27.
    Roman, L. J., Eggleston, A. K., and Kowalczykowski, S. C. (1992) Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. J. Biol. Chem. 267, 4207–4214.PubMedGoogle Scholar
  28. 28.
    Weiss, B. (1981) Exodeoxyribonucleases of Escherichia coli, in The Enzymes, vol. XIV (Boyer, P. D., ed.), Academic Press, New York, pp. 203–231.Google Scholar
  29. 29.
    Anderson, D. G. and Kowalczykowski, S. C. (1997) The recombination hot spot κ is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 11, 571–581.PubMedCrossRefGoogle Scholar
  30. 30.
    McKittrick, N. H. and Smith, G. R. (1989) Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria. J. Mol. Biol. 210, 485–495.PubMedCrossRefGoogle Scholar
  31. 31.
    Sourice, S., Biaudet, V., El Karoui, M., et al. (1998) Identification of the Chi site of Haemophilus influenzae as several sequences related to the Escherichia coli Chi site. Mol. Microbiol. 27, [pp1021-1029].Google Scholar
  32. 32.
    Cleaver, J. E. and Boyer, H. W. (1972) Solubility and dialysis limits of DNA oligonucleotides. Biochim. Biophys. Acta 262, 116–124.PubMedGoogle Scholar
  33. 33.
    Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  34. 34.
    Sasaki, M., Fujiyoshi, T., Shimada, K., and Takagi, Y. (1982) Fine structure of the recB and recC gene region of Escherichia coli. Biochem. Biophys. Res. Commun. 109, 414–422.PubMedCrossRefGoogle Scholar
  35. 35.
    Korangy, F. and Julin, D. A. (1992) Alteration by site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of the Escherichia coli RecBCD enzyme. J. Biol. Chem. 267, 1727–1732.PubMedGoogle Scholar
  36. 36.
    Mead, D. A., Szczesna-Skorupa, E., and Kemper, B. (1986) Single-strand DNA “blue” T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1, 67–74.PubMedCrossRefGoogle Scholar
  37. 37.
    Amundsen, S. K., Taylor, A. F., Chaudhury, A. M., and Smith, G. R. (1986) recD: the gene for an essential third subunit of exonuclease V. Proc. Natl. Acad. Sci. USA 83, 5558–5562.PubMedCrossRefGoogle Scholar
  38. 38.
    Julin, D. A. and Lehman, I. R. (1987) Photoaffinity labelling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5′-triphosphate. J. Biol. Chem. 262, 9044–9051.PubMedGoogle Scholar
  39. 39.
    Finch, P. W., Storey, A., Brown, K., et al. (1986) Complete nucleotide sequence of recD, the structural gene for the α subunit of Exonuclease V of Escherichia coli. Nucleic Acids Res. 14, 8583–8594.PubMedCrossRefGoogle Scholar
  40. 40.
    Eichler, D. C. and Lehman, I. R. (1977) On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. J. Biol. Chem. 252, 499–503.PubMedGoogle Scholar
  41. 41.
    Dykstra, C. C., Palas, K. M., and Kushner, S. R. (1984) Purification and characterization of Exonuclease V from Escherichia coli K-12. Cold Spring Harbor Symp. Quant. Biol. XLIX, 463–467.Google Scholar
  42. 42.
    Masterson, C., Boehmer, P. E., McDonald, F., et al. (1992) Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. J. Biol. Chem. 267, 13,564–13,572.PubMedGoogle Scholar
  43. 43.
    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.PubMedCrossRefGoogle Scholar
  44. 44.
    O’Sullivan, W. J. and Smithers, G. W. (1979) Stability constants for biologically important metal-ligand complexes. Methods Enzymol. 63, 294–336.PubMedCrossRefGoogle Scholar
  45. 45.
    Weast, R. C., ed. (1987) CRC Handbook of Chemistry and Physics. CRC, Boca Raton, FL, p. D–162.Google Scholar
  46. 46.
    Kornberg, A. and Baker, T. A. (1991) DNA Replication, 2nd Ed., W. H. Freeman, New York, p. 89.Google Scholar
  47. 47.
    Rodriguez, R. L. and Tait, R. C. (1983) Recombinant DNA Techniques: An Introduction, Benjamin/Cummings, Menlo Park, CA, p. 43.Google Scholar
  48. 48.
    Halford, S. E. and Goodall, A. J. (1988) Modes of DNA cleavage by the EcoRV restriction endonuclease. Biochemistry 27, 1771–1777.PubMedCrossRefGoogle Scholar
  49. 49.
    Karu, A. E., MacKay, V., Goldmark, P. J., and Linn, S. (1973) The recBC deoxyribonuclease of Escherichia coli K-12. J. Biol. Chem. 248, 4874–4884.PubMedGoogle Scholar
  50. 50.
    Hsieh, S. and Julin, D. A. (1992) Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein of Escherichia coli. Nucleic Acids Res. 20, 5647–5653.PubMedCrossRefGoogle Scholar
  51. 51.
    Roman, L. J. and Kowalczykowski, S. C. (1989) Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA. Biochemistry 28, 2873–2881.PubMedCrossRefGoogle Scholar
  52. 52.
    Korangy, F. and Julin, D. A. (1992) Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli. J. Biol. Chem. 267, 1733–1740.PubMedGoogle Scholar

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© Humana Press Inc. 2000

Authors and Affiliations

  • Douglas A. Julin
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of MarylandCollege Park

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