Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jul 15;25(14):2841–2846. doi: 10.1093/nar/25.14.2841

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

C Finta 1, A Kiss 1
PMCID: PMC146830  PMID: 9207033

Abstract

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.

Full Text

The Full Text of this article is available as a PDF (261.8 KB).

Selected References

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

  1. Balganesh T. S., Reiners L., Lauster R., Noyer-Weidner M., Wilke K., Trautner T. A. Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions. EMBO J. 1987 Nov;6(11):3543–3549. doi: 10.1002/j.1460-2075.1987.tb02681.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chen L., MacMillan A. M., Chang W., Ezaz-Nikpay K., Lane W. S., Verdine G. L. Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. Biochemistry. 1991 Nov 19;30(46):11018–11025. doi: 10.1021/bi00110a002. [DOI] [PubMed] [Google Scholar]
  3. Dixon W. J., Hayes J. J., Levin J. R., Weidner M. F., Dombroski B. A., Tullius T. D. Hydroxyl radical footprinting. Methods Enzymol. 1991;208:380–413. doi: 10.1016/0076-6879(91)08021-9. [DOI] [PubMed] [Google Scholar]
  4. Dubey A. K., Roberts R. J. Sequence-specific DNA binding by the MspI DNA methyltransferase. Nucleic Acids Res. 1992 Jun 25;20(12):3167–3173. doi: 10.1093/nar/20.12.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Klimasauskas S., Nelson J. L., Roberts R. J. The sequence specificity domain of cytosine-C5 methylases. Nucleic Acids Res. 1991 Nov 25;19(22):6183–6190. doi: 10.1093/nar/19.22.6183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kumar S., Cheng X., Klimasauskas S., Mi S., Posfai J., Roberts R. J., Wilson G. G. The DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 1994 Jan 11;22(1):1–10. doi: 10.1093/nar/22.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lauster R., Trautner T. A., Noyer-Weidner M. Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains. J Mol Biol. 1989 Mar 20;206(2):305–312. doi: 10.1016/0022-2836(89)90480-4. [DOI] [PubMed] [Google Scholar]
  10. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  11. O'Gara M., Klimasauskas S., Roberts R. J., Cheng X. Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaL methyltransferase-DNA-AdoHcy complexes. J Mol Biol. 1996 Sep 6;261(5):634–645. doi: 10.1006/jmbi.1996.0489. [DOI] [PubMed] [Google Scholar]
  12. O'Gara M., Roberts R. J., Cheng X. A structural basis for the preferential binding of hemimethylated DNA by HhaI DNA methyltransferase. J Mol Biol. 1996 Nov 8;263(4):597–606. doi: 10.1006/jmbi.1996.0601. [DOI] [PubMed] [Google Scholar]
  13. Papavassiliou A. G. Chemical nucleases as probes for studying DNA-protein interactions. Biochem J. 1995 Jan 15;305(Pt 2):345–357. doi: 10.1042/bj3050345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pósfai G., Kiss A., Erdei S., Pósfai J., Venetianer P. Structure of the Bacillus sphaericus R modification methylase gene. J Mol Biol. 1983 Nov 5;170(3):597–610. doi: 10.1016/s0022-2836(83)80123-5. [DOI] [PubMed] [Google Scholar]
  15. Reinisch K. M., Chen L., Verdine G. L., Lipscomb W. N. The crystal structure of HaeIII methyltransferase convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell. 1995 Jul 14;82(1):143–153. doi: 10.1016/0092-8674(95)90060-8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sigman D. S., Kuwabara M. D., Chen C. H., Bruice T. W. Nuclease activity of 1,10-phenanthroline-copper in study of protein-DNA interactions. Methods Enzymol. 1991;208:414–433. doi: 10.1016/0076-6879(91)08022-a. [DOI] [PubMed] [Google Scholar]
  19. Slatko B. E., Croft R., Moran L. S., Wilson G. G. Cloning and analysis of the HaeIII and HaeII methyltransferase genes. Gene. 1988 Dec 25;74(1):45–50. doi: 10.1016/0378-1119(88)90248-x. [DOI] [PubMed] [Google Scholar]
  20. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  21. Szczelkun M. D., Jones H., Connolly B. A. Probing the protein-DNA interface of the EcoRV modification methyltransferase bound to its recognition sequence, GATATC. Biochemistry. 1995 Aug 29;34(34):10734–10743. doi: 10.1021/bi00034a005. [DOI] [PubMed] [Google Scholar]
  22. Wu J. C., Santi D. V. Kinetic and catalytic mechanism of HhaI methyltransferase. J Biol Chem. 1987 Apr 5;262(10):4778–4786. [PubMed] [Google Scholar]

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

RESOURCES