• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Feb 1, 1997; 25(3): 503–511.
PMCID: PMC146478

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.


Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.

Full Text

The Full Text of this article is available as a PDF (137K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Wilson GG, Murray NE. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. [PubMed]
  • Bickle TA, Krüger DH. Biology of DNA restriction. Microbiol Rev. 1993 Jun;57(2):434–450. [PMC free article] [PubMed]
  • Hubacek J, Glover SW. Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coli. J Mol Biol. 1970 May 28;50(1):111–127. [PubMed]
  • Taylor I, Patel J, Firman K, Kneale G. Purification and biochemical characterisation of the EcoR124 type I modification methylase. Nucleic Acids Res. 1992 Jan 25;20(2):179–186. [PMC free article] [PubMed]
  • Dryden DT, Cooper LP, Murray NE. Purification and characterization of the methyltransferase from the type 1 restriction and modification system of Escherichia coli K12. J Biol Chem. 1993 Jun 25;268(18):13228–13236. [PubMed]
  • Murray NE, Daniel AS, Cowan GM, Sharp PM. Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes. Mol Microbiol. 1993 Jul;9(1):133–143. [PubMed]
  • Yuan R. Structure and mechanism of multifunctional restriction endonucleases. Annu Rev Biochem. 1981;50:285–319. [PubMed]
  • Yuan R, Bickle TA, Ebbers W, Brack C. Multiple steps in DNA recognition by restriction endonuclease from E. coli K. Nature. 1975 Aug 14;256(5518):556–560. [PubMed]
  • Studier FW, Bandyopadhyay PK. Model for how type I restriction enzymes select cleavage sites in DNA. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4677–4681. [PMC free article] [PubMed]
  • Endlich B, Linn S. The DNA restriction endonuclease of Escherichia coli B. II. Further studies of the structure of DNA intermediates and products. J Biol Chem. 1985 May 10;260(9):5729–5738. [PubMed]
  • Yuan R, Heywood J, Meselson M. ATP hydrolysis by restriction endonuclease from E. coli K. Nat New Biol. 1972 Nov 8;240(97):42–43. [PubMed]
  • Price C, Shepherd JC, Bickle TA. DNA recognition by a new family of type I restriction enzymes: a unique relationship between two different DNA specificities. EMBO J. 1987 May;6(5):1493–1497. [PMC free article] [PubMed]
  • Piekarowicz A, Goguen JD. The DNA sequence recognized by the EcoDXX1 restriction endonuclease. Eur J Biochem. 1986 Jan 15;154(2):295–298. [PubMed]
  • Tyndall C, Meister J, Bickle TA. The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene. J Mol Biol. 1994 Apr 1;237(3):266–274. [PubMed]
  • Price C, Lingner J, Bickle TA, Firman K, Glover SW. Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. J Mol Biol. 1989 Jan 5;205(1):115–125. [PubMed]
  • Abadjieva A, Patel J, Webb M, Zinkevich V, Firman K. A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity. Nucleic Acids Res. 1993 Sep 25;21(19):4435–4443. [PMC free article] [PubMed]
  • Meister J, MacWilliams M, Hübner P, Jütte H, Skrzypek E, Piekarowicz A, Bickle TA. Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition. EMBO J. 1993 Dec;12(12):4585–4591. [PMC free article] [PubMed]
  • Amitsur M, Morad I, Chapman-Shimshoni D, Kaufmann G. HSD restriction-modification proteins partake in latent anticodon nuclease. EMBO J. 1992 Aug;11(8):3129–3134. [PMC free article] [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Firman K, Price C, Glover SW. The EcoR124 and EcoR124/3 restriction and modification systems: cloning the genes. Plasmid. 1985 Nov;14(3):224–234. [PubMed]
  • Amann E, Ochs B, Abel KJ. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. [PubMed]
  • Abadjieva A, Webb M, Patel J, Zinkevich V, Firman K. Deletions within the DNA recognition subunit of M.EcoR124I that identify a region involved in protein-protein interactions between HsdS and HsdM. J Mol Biol. 1994 Aug 5;241(1):35–43. [PubMed]
  • Chan KM, Delfert D, Junger KD. A direct colorimetric assay for Ca2+ -stimulated ATPase activity. Anal Biochem. 1986 Sep;157(2):375–380. [PubMed]
  • Janscak P, Abadjieva A, Firman K. The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties. J Mol Biol. 1996 Apr 19;257(5):977–991. [PubMed]
  • Meisel A, Mackeldanz P, Bickle TA, Krüger DH, Schroeder C. Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis. EMBO J. 1995 Jun 15;14(12):2958–2966. [PMC free article] [PubMed]
  • Endlich B, Linn S. The DNA restriction endonuclease of Escherichia coli B. I. Studies of the DNA translocation and the ATPase activities. J Biol Chem. 1985 May 10;260(9):5720–5728. [PubMed]
  • Yuan R, Hamilton DL, Burckhardt J. DNA translocation by the restriction enzyme from E. coli K. Cell. 1980 May;20(1):237–244. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • JACOB F, WOLLMAN EL. Etude génétique d'un bactériophage tempéré d'Escherichia coli. l. Le système genétique du bactériophage. Ann Inst Pasteur (Paris) 1954 Dec;87(6):653–673. [PubMed]
  • Mead DA, Szczesna-Skorupa E, Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. [PubMed]
  • Patel J, Taylor I, Dutta CF, Kneale G, Firman K. High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124. Gene. 1992 Mar 1;112(1):21–27. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...