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. 1996 Dec 15;24(24):4853–4858. doi: 10.1093/nar/24.24.4853

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

D R Mernagh 1, G G Kneale 1
PMCID: PMC146333  PMID: 9016653

Abstract

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.

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Selected References

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  1. Bickle T. A., Krüger D. H. Biology of DNA restriction. Microbiol Rev. 1993 Jun;57(2):434–450. doi: 10.1128/mr.57.2.434-450.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cheng X., Kumar S., Posfai J., Pflugrath J. W., Roberts R. J. Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine. Cell. 1993 Jul 30;74(2):299–307. doi: 10.1016/0092-8674(93)90421-l. [DOI] [PubMed] [Google Scholar]
  3. Cowan G. M., Gann A. A., Murray N. E. Conservation of complex DNA recognition domains between families of restriction enzymes. Cell. 1989 Jan 13;56(1):103–109. doi: 10.1016/0092-8674(89)90988-4. [DOI] [PubMed] [Google Scholar]
  4. Dryden D. T., Cooper L. P., Murray N. E. 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] [Google Scholar]
  5. Fairall L., Rhodes D. A new approach to the analysis of DNase I footprinting data and its application to the TFIIIA/5S DNA complex. Nucleic Acids Res. 1992 Sep 25;20(18):4727–4731. doi: 10.1093/nar/20.18.4727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fuller-Pace F. V., Cowan G. M., Murray N. E. EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli. J Mol Biol. 1985 Nov 5;186(1):65–75. doi: 10.1016/0022-2836(85)90257-8. [DOI] [PubMed] [Google Scholar]
  7. Fuller-Pace F. V., Murray N. E. Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9368–9372. doi: 10.1073/pnas.83.24.9368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gubler M., Braguglia D., Meyer J., Piekarowicz A., Bickle T. A. Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes. EMBO J. 1992 Jan;11(1):233–240. doi: 10.1002/j.1460-2075.1992.tb05046.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kannan P., Cowan G. M., Daniel A. S., Gann A. A., Murray N. E. Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes. J Mol Biol. 1989 Oct 5;209(3):335–344. doi: 10.1016/0022-2836(89)90001-6. [DOI] [PubMed] [Google Scholar]
  10. Klimasauskas S., Kumar S., Roberts R. J., Cheng X. HhaI methyltransferase flips its target base out of the DNA helix. Cell. 1994 Jan 28;76(2):357–369. doi: 10.1016/0092-8674(94)90342-5. [DOI] [PubMed] [Google Scholar]
  11. Kneale G. G. A symmetrical model for the domain structure of type I DNA methyltransferases. J Mol Biol. 1994 Oct 14;243(1):1–5. doi: 10.1006/jmbi.1994.1624. [DOI] [PubMed] [Google Scholar]
  12. Labahn J., Granzin J., Schluckebier G., Robinson D. P., Jack W. E., Schildkraut I., Saenger W. Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10957–10961. doi: 10.1073/pnas.91.23.10957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lavoie B. D., Chan B. S., Allison R. G., Chaconas G. Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J. 1991 Oct;10(10):3051–3059. doi: 10.1002/j.1460-2075.1991.tb07856.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leblanc B., Moss T. DNase I footprinting. Methods Mol Biol. 1994;30:1–10. doi: 10.1385/0-89603-256-6:1. [DOI] [PubMed] [Google Scholar]
  15. Murray N. E., Gough J. A., Suri B., Bickle T. A. Structural homologies among type I restriction-modification systems. EMBO J. 1982;1(5):535–539. doi: 10.1002/j.1460-2075.1982.tb01205.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Patel J., Taylor I., Dutta C. F., 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. doi: 10.1016/0378-1119(92)90298-4. [DOI] [PubMed] [Google Scholar]
  17. Powell L. M., Dryden D. T., Willcock D. F., Pain R. H., Murray N. E. DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine. J Mol Biol. 1993 Nov 5;234(1):60–71. doi: 10.1006/jmbi.1993.1563. [DOI] [PubMed] [Google Scholar]
  18. Powell L. M., Murray N. E. S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase. Nucleic Acids Res. 1995 Mar 25;23(6):967–974. doi: 10.1093/nar/23.6.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Price C., Pripfl T., Bickle T. A. EcoR124 and EcoR124/3: the first members of a new family of type I restriction and modification systems. Eur J Biochem. 1987 Aug 17;167(1):111–115. doi: 10.1111/j.1432-1033.1987.tb13310.x. [DOI] [PubMed] [Google Scholar]
  20. Renbaum P., Razin A. Footprint analysis of M.Sssl and M.Hhal methyltransferases reveals extensive interactions with the substrate DNA backbone. J Mol Biol. 1995 Apr 21;248(1):19–26. doi: 10.1006/jmbi.1995.0199. [DOI] [PubMed] [Google Scholar]
  21. Schickor P., Heumann H. Hydroxyl radical footprinting. Methods Mol Biol. 1994;30:21–32. doi: 10.1385/0-89603-256-6:21. [DOI] [PubMed] [Google Scholar]
  22. Schluckebier G., O'Gara M., Saenger W., Cheng X. Universal catalytic domain structure of AdoMet-dependent methyltransferases. J Mol Biol. 1995 Mar 17;247(1):16–20. doi: 10.1006/jmbi.1994.0117. [DOI] [PubMed] [Google Scholar]
  23. Suri B., Bickle T. A. EcoA: the first member of a new family of type I restriction modification systems. Gene organization and enzymatic activities. J Mol Biol. 1985 Nov 5;186(1):77–85. doi: 10.1016/0022-2836(85)90258-x. [DOI] [PubMed] [Google Scholar]
  24. Taylor I. A., Davis K. G., Watts D., Kneale G. G. DNA-binding induces a major structural transition in a type I methyltransferase. EMBO J. 1994 Dec 1;13(23):5772–5778. doi: 10.1002/j.1460-2075.1994.tb06915.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Taylor I. A., Webb M., Kneale G. G. Surface labelling of the type I methyltransferase M.EcoR124I reveals lysine residues critical for DNA binding. J Mol Biol. 1996 Apr 26;258(1):62–73. doi: 10.1006/jmbi.1996.0234. [DOI] [PubMed] [Google Scholar]
  26. 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. doi: 10.1093/nar/20.2.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taylor I., Watts D., Kneale G. Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I. Nucleic Acids Res. 1993 Oct 25;21(21):4929–4935. doi: 10.1093/nar/21.21.4929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tyndall C., Meister J., Bickle T. A. 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. doi: 10.1006/jmbi.1994.1230. [DOI] [PubMed] [Google Scholar]
  30. Webb M., Taylor I. A., Firman K., Kneale G. G. Probing the domain structure of the type IC DNA methyltransferase M.EcoR124I by limited proteolysis. J Mol Biol. 1995 Jul 7;250(2):181–190. doi: 10.1006/jmbi.1995.0369. [DOI] [PubMed] [Google Scholar]
  31. Wilson G. G., Murray N. E. Restriction and modification systems. Annu Rev Genet. 1991;25:585–627. doi: 10.1146/annurev.ge.25.120191.003101. [DOI] [PubMed] [Google Scholar]
  32. Yang C. C., Nash H. A. The interaction of E. coli IHF protein with its specific binding sites. Cell. 1989 Jun 2;57(5):869–880. doi: 10.1016/0092-8674(89)90801-5. [DOI] [PubMed] [Google Scholar]

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