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. 1996 Mar 15;15(6):1443–1450.

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

C Lange 1, C Wild 1, T A Trautner 1
PMCID: PMC450049  PMID: 8635477

Abstract

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.

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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. Behrens B., Noyer-Weidner M., Pawlek B., Lauster R., Balganesh T. S., Trautner T. A. Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages. EMBO J. 1987 Apr;6(4):1137–1142. doi: 10.1002/j.1460-2075.1987.tb04869.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buhk H. J., Behrens B., Tailor R., Wilke K., Prada J. J., Günthert U., Noyer-Weidner M., Jentsch S., Trautner T. A. Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR. Gene. 1984 Jul-Aug;29(1-2):51–61. doi: 10.1016/0378-1119(84)90165-3. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Feil R., Charlton J., Bird A. P., Walter J., Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing. Nucleic Acids Res. 1994 Feb 25;22(4):695–696. doi: 10.1093/nar/22.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frommer M., McDonald L. E., Millar D. S., Collis C. M., Watt F., Grigg G. W., Molloy P. L., Paul C. L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827–1831. doi: 10.1073/pnas.89.5.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuller-Pace F. V., Bullas L. R., Delius H., Murray N. E. Genetic recombination can generate altered restriction specificity. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6095–6099. doi: 10.1073/pnas.81.19.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  9. Gann A. A., Campbell A. J., Collins J. F., Coulson A. F., Murray N. E. Reassortment of DNA recognition domains and the evolution of new specificities. Mol Microbiol. 1987 Jul;1(1):13–22. doi: 10.1111/j.1365-2958.1987.tb00521.x. [DOI] [PubMed] [Google Scholar]
  10. Günthert U., Lauster R., Reiners L. Multispecific DNA methyltransferases from Bacillus subtilis phages. Properties of wild-type and various mutant enzymes with altered DNA affinity. Eur J Biochem. 1986 Sep 15;159(3):485–492. doi: 10.1111/j.1432-1033.1986.tb09912.x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  16. Lange C., Jugel A., Walter J., Noyer-Weidner M., Trautner T. A. 'Pseudo' domains in phage-encoded DNA methyltransferases. Nature. 1991 Aug 15;352(6336):645–648. doi: 10.1038/352645a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Nagaraja V., Shepherd J. C., Bickle T. A. A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity. Nature. 1985 Jul 25;316(6026):371–372. doi: 10.1038/316371a0. [DOI] [PubMed] [Google Scholar]
  20. Noyer-Weidner M., Walter J., Terschüren P. A., Chai S., Trautner T. A. M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases. Nucleic Acids Res. 1994 Oct 11;22(20):4066–4072. doi: 10.1093/nar/22.20.4066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ohmori H., Tomizawa J. I., Maxam A. M. Detection of 5-methylcytosine in DNA sequences. Nucleic Acids Res. 1978 May;5(5):1479–1485. doi: 10.1093/nar/5.5.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pósfai J., Bhagwat A. S., Pósfai G., Roberts R. J. Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res. 1989 Apr 11;17(7):2421–2435. doi: 10.1093/nar/17.7.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tran-Betcke A., Behrens B., Noyer-Weidner M., Trautner T. A. DNA methyltransferase genes of Bacillus subtilis phages: comparison of their nucleotide sequences. Gene. 1986;42(1):89–96. doi: 10.1016/0378-1119(86)90153-8. [DOI] [PubMed] [Google Scholar]
  26. Trautner T. A., Balganesh T. S., Pawlek B. Chimeric multispecific DNA methyltransferases with novel combinations of target recognition. Nucleic Acids Res. 1988 Jul 25;16(14A):6649–6658. doi: 10.1093/nar/16.14.6649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trautner T. A., Pawlek B., Behrens B., Willert J. Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases. EMBO J. 1996 Mar 15;15(6):1434–1442. [PMC free article] [PubMed] [Google Scholar]
  28. Walter J., Trautner T. A., Noyer-Weidner M. High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules. EMBO J. 1992 Dec;11(12):4445–4450. doi: 10.1002/j.1460-2075.1992.tb05545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wilke K., Rauhut E., Noyer-Weidner M., Lauster R., Pawlek B., Behrens B., Trautner T. A. Sequential order of target-recognizing domains in multispecific DNA-methyltransferases. EMBO J. 1988 Aug;7(8):2601–2609. doi: 10.1002/j.1460-2075.1988.tb03110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]

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