Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2002 Jun;161(2):483–492. doi: 10.1093/genetics/161.2.483

A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme.

Susan K Amundsen 1, Andrew F Taylor 1, Gerald R Smith 1
PMCID: PMC1462144  PMID: 12072448

Abstract

The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange. Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity. Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations. Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain. RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends. The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.

Full Text

The Full Text of this article is available as a PDF (227.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amundsen S. K., Neiman A. M., Thibodeaux S. M., Smith G. R. Genetic dissection of the biochemical activities of RecBCD enzyme. Genetics. 1990 Sep;126(1):25–40. doi: 10.1093/genetics/126.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amundsen S. K., Taylor A. F., Chaudhury A. M., Smith G. R. recD: the gene for an essential third subunit of exonuclease V. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5558–5562. doi: 10.1073/pnas.83.15.5558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amundsen S. K., Taylor A. F., Smith G. R. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7399–7404. doi: 10.1073/pnas.130192397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson D. G., Churchill J. J., Kowalczykowski S. C. A single mutation, RecB(D1080A,) eliminates RecA protein loading but not Chi recognition by RecBCD enzyme. J Biol Chem. 1999 Sep 17;274(38):27139–27144. doi: 10.1074/jbc.274.38.27139. [DOI] [PubMed] [Google Scholar]
  5. Anderson D. G., Kowalczykowski S. C. The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner. Cell. 1997 Jul 11;90(1):77–86. doi: 10.1016/s0092-8674(00)80315-3. [DOI] [PubMed] [Google Scholar]
  6. Arnold D. A., Handa N., Kobayashi I., Kowalczykowski S. C. A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi. J Mol Biol. 2000 Jul 14;300(3):469–479. doi: 10.1006/jmbi.2000.3861. [DOI] [PubMed] [Google Scholar]
  7. Arnold D. A., Kowalczykowski S. C. Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme. J Biol Chem. 2000 Apr 21;275(16):12261–12265. doi: 10.1074/jbc.275.16.12261. [DOI] [PubMed] [Google Scholar]
  8. Boehmer P. E., Emmerson P. T. The RecB subunit of the Escherichia coli RecBCD enzyme couples ATP hydrolysis to DNA unwinding. J Biol Chem. 1992 Mar 5;267(7):4981–4987. [PubMed] [Google Scholar]
  9. Chaudhury A. M., Smith G. R. A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7850–7854. doi: 10.1073/pnas.81.24.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Churchill J. J., Anderson D. G., Kowalczykowski S. C. The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation. Genes Dev. 1999 Apr 1;13(7):901–911. doi: 10.1101/gad.13.7.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dixon D. A., Kowalczykowski S. C. Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell. 1991 Jul 26;66(2):361–371. doi: 10.1016/0092-8674(91)90625-9. [DOI] [PubMed] [Google Scholar]
  12. Dixon D. A., Kowalczykowski S. C. The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell. 1993 Apr 9;73(1):87–96. doi: 10.1016/0092-8674(93)90162-j. [DOI] [PubMed] [Google Scholar]
  13. Eichler D. C., Lehman I. R. On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. J Biol Chem. 1977 Jan 25;252(2):499–503. [PubMed] [Google Scholar]
  14. Finch P. W., Storey A., Brown K., Hickson I. D., Emmerson P. T. Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli. Nucleic Acids Res. 1986 Nov 11;14(21):8583–8594. doi: 10.1093/nar/14.21.8583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finch P. W., Wilson R. E., Brown K., Hickson I. D., Tomkinson A. E., Emmerson P. T. Complete nucleotide sequence of the Escherichia coli recC gene and of the thyA-recC intergenic region. Nucleic Acids Res. 1986 Jun 11;14(11):4437–4451. doi: 10.1093/nar/14.11.4437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Handa N., Ohashi S., Kusano K., Kobayashi I. Chi-star, a chi-related 11-mer sequence partially active in an E. coli recC1004 strain. Genes Cells. 1997 Aug;2(8):525–536. doi: 10.1046/j.1365-2443.1997.1410339.x. [DOI] [PubMed] [Google Scholar]
  17. Hickson I. D., Robson C. N., Atkinson K. E., Hutton L., Emmerson P. T. Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins. J Biol Chem. 1985 Jan 25;260(2):1224–1229. [PubMed] [Google Scholar]
  18. Howard-Flanders P., Theriot L. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics. 1966 Jun;53(6):1137–1150. doi: 10.1093/genetics/53.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Julin D. A., Lehman I. R. Photoaffinity labeling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5'-triphosphate. J Biol Chem. 1987 Jul 5;262(19):9044–9051. [PubMed] [Google Scholar]
  20. Korangy F., Julin D. A. A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli. J Biol Chem. 1992 Feb 15;267(5):3088–3095. [PubMed] [Google Scholar]
  21. Korangy F., Julin D. A. Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. Biochemistry. 1993 May 11;32(18):4873–4880. doi: 10.1021/bi00069a024. [DOI] [PubMed] [Google Scholar]
  22. Kowalczykowski S. C., Dixon D. A., Eggleston A. K., Lauder S. D., Rehrauer W. M. Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev. 1994 Sep;58(3):401–465. doi: 10.1128/mr.58.3.401-465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  24. Oliver D. B., Goldberg E. B. Protection of parental T4 DNA from a restriction exonuclease by the product of gene 2. J Mol Biol. 1977 Nov;116(4):877–881. doi: 10.1016/0022-2836(77)90276-5. [DOI] [PubMed] [Google Scholar]
  25. Ponticelli A. S., Schultz D. W., Taylor A. F., Smith G. R. Chi-dependent DNA strand cleavage by RecBC enzyme. Cell. 1985 May;41(1):145–151. doi: 10.1016/0092-8674(85)90069-8. [DOI] [PubMed] [Google Scholar]
  26. Rinken R., Wackernagel W. Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli. J Bacteriol. 1992 Feb;174(4):1172–1178. doi: 10.1128/jb.174.4.1172-1178.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schultz D. W., Taylor A. F., Smith G. R. Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity. J Bacteriol. 1983 Aug;155(2):664–680. doi: 10.1128/jb.155.2.664-680.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith G. R. Homologous recombination near and far from DNA breaks: alternative roles and contrasting views. Annu Rev Genet. 2001;35:243–274. doi: 10.1146/annurev.genet.35.102401.090509. [DOI] [PubMed] [Google Scholar]
  29. Stahl F. W., Thomason L. C., Siddiqi I., Stahl M. M. Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli. Genetics. 1990 Nov;126(3):519–533. doi: 10.1093/genetics/126.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Taylor A. F., Schultz D. W., Ponticelli A. S., Smith G. R. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell. 1985 May;41(1):153–163. doi: 10.1016/0092-8674(85)90070-4. [DOI] [PubMed] [Google Scholar]
  31. Taylor A. F., Smith G. R. Monomeric RecBCD enzyme binds and unwinds DNA. J Biol Chem. 1995 Oct 13;270(41):24451–24458. doi: 10.1074/jbc.270.41.24451. [DOI] [PubMed] [Google Scholar]
  32. Taylor A. F., Smith G. R. RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5226–5230. doi: 10.1073/pnas.89.12.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Taylor A. F., Smith G. R. Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits. Genes Dev. 1999 Apr 1;13(7):890–900. doi: 10.1101/gad.13.7.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tomizawa J., Ogawa H. Structural genes of ATP-dependent deoxyribonuclease of Escherichia coli. Nat New Biol. 1972 Sep 6;239(88):14–16. doi: 10.1038/newbio239014a0. [DOI] [PubMed] [Google Scholar]
  35. Willetts N. S., Clark A. J., Low B. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol. 1969 Jan;97(1):244–249. doi: 10.1128/jb.97.1.244-249.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yu M., Souaya J., Julin D. A. Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. J Mol Biol. 1998 Nov 6;283(4):797–808. doi: 10.1006/jmbi.1998.2127. [DOI] [PubMed] [Google Scholar]
  37. Yu M., Souaya J., Julin D. A. The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):981–986. doi: 10.1073/pnas.95.3.981. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES