Research - Institute of Biochemistry - Synthetic and Systems Biology Unit - Laboratory of DNA-Protein Interactions

Antal KISS
scientific adviser

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Mihály KONCZ scientific administrator
Pál ALBERT Ph.D. student
Nikolett ZSIBRITA Ph.D. student
Zita PLETL laboratory assistant

DNA METHYLTRANSFERASES: TOOLS FOR GENOME RESEARCH

The prokaryotic DNA methyltransferase M.SssI shares the specificity (5’-CG) of mammalian DNA methyltransferases qualifying it as a model system and a research tool for the study of many aspects of DNA methylation in higher eukaryotes. Our goal is to develop M.SssI into a targetable DNA methyltransferase that can selectively methylate predetermined sites in genomic DNA. Such programmable methyltransferases would be excellent research tools for studying DNA methylation in higher eukaryotes and can lead to new therapeutic approaches.


Targeted DNA methylation

Cytosine-5 methylation of 5’-CG-sites in the genome is an important component of epigenetic regulation in higher eukaryotes. DNA methylation plays key roles in a number of biological phenomena such as X-chromosome inactivation, genomic imprinting and inactivation of genomic parazites. The pattern of genomic DNA methylation (the methylome) correlates with the function of the cell and with tissue-specific gene expression. There is an increasing body of evidence supporting the role of aberrant DNA methylation in the pathogenesis of several diseases including cancer.

Methylation of CG sites in promoters of genes is thought to lead to gene silencing. This raises the possibility of silencing selected genes by targeting methylation to their promoters. Silencing of genes by directed methylation would be an excellent research tool in the study of the biological role of DNA methylation and could lead to new therapeutic approaches in the future. The general approach to directed DNA methylation pioneered by Xu and Bestor (Nat. Genet. 17: 376-378, 1997) has been to link a DNA methyltransferase with a targeting domain, typically a zinc finger protein engineered to bind with high specificity to a DNA sequence located adjacently to the targeted CG site. Although in a few cases considerable level of targeting specificity has been demonstrated, this technique is not widely used. One of the reasons can be that with the available approaches methylation selectivity is not sufficiently high, i.e. non-targeted CG sites are also methylated at detectable frequency. Off-target methylation is not surprising because DNA methyltransferases have affinity to their substrate site even when they are fused to a targeting domain. Another limitation of some of the published methods is that they were developed for DNA methyltransferases, which – due to their specificity (e.g. CCGG) – can methylate only a subset of CG sites.

Our goal is to develop a technique, which can be used to methylate CGs in any sequence context with high specificity. We use the bacterial DNA methyltransferase M.SssI, which has the same sequence specificity as the eukaryotic enzymes (CG). We plan to construct M.SssI variants, which methylate only when bound, via the zinc finger targeting domain, in the vicinity of the targeted CG site. Mutant variants of M.SssI are genetically fused to zinc finger proteins and specificity of targeted methylation is tested in vivo in E. coli.

In a related approach to targeted DNA methylation we try to use, instead of zinc fingers, triplex-forming-oligonucleotides as targeting domain. This latter project is carried out in collaboration with Elmar Weinhold (RWTH Aachen University, Aachen, Germany) and Marianne Rots (Groningen University Medical Center, Groningen, The Netherlands).

Selected publications

Heitman, J., Ivanenko, T. and Kiss, A. (1999). DNA nicks inflicted by restriction endonucleases are repaired by a RecA- and RecB-dependent pathway in Escherichia coli. Mol. Microbiol. 33: 1141-1151.

Raskó, T., Finta, C. and Kiss, A. (2000). DNA bending induced by DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 28: 3083-3091.

Simoncsits, A., Tjörnhammar, M.-L., Raskó, T., Kiss, A. and Pongor, S. (2001). Covalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease. J. Mol. Biol. 309: 89-97.

Kiss, A., Pósfai, G., Zsurka, G., Raskó, T. and Venetianer, P. (2001). Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 29: 3188-3194.

Roberts, R.J., Belfort, M., Bestor, T., Bhagwat, A.S., Bickle, T.A., Bitinaite, J., Blumenthal, R.M., Degtyarev, S.K., Dryden, D.T.F., Dybvig, K., Firman, K., Gromova, E.S., Gumport, R.I., Halford, S.E., Hattman, S., Heitman, J., Hornby, D.P., Janulaitis, A., Jeltsch, A., Josephsen, J., Kiss, A., Klaenhammer, T.R., Kobayashi, I., Kong, H., Krüger, D.H., Lacks, S., Marinus, M.G., Miyahara, M., Morgan, R.D., Murray, N.E., Nagaraja, V., Piekarowicz, A., Pingoud, A., Raleigh, E., Rao, D.N., Reich, N., Repin, V.E., Selker, E.U., Shaw, P.-C., Stein, D.C., Stoddard, B.L., Szybalski, W., Trautner, T.A., Van Etten, J.L., Vitor, J.M.B., Wilson, G.G. and Xu, S. (2003). A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31: 1805-1812.

Tímár, E., Groma, G., Kiss, A. and Venetianer, P. (2004). Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution. Nucleic Acids Res. 32: 3898-3903.

Rathert, P., Raskó, T., Roth, M., Ślaska-Kiss, K., Pingoud, A., Kiss, A. and Jeltsch, A. (2007). Reversible inactivation of the CG-specific SssI DNA-(cytosine-C5)-methyltransferase with a photocleavable protection group. ChemBioChem 8: 202-207.

Kiss, A. and Weinhold, E. (2008). Functional reassembly of split enzymes on-site: A novel approach for highly sequence-specific targeted DNA methylation. ChemBioChem 9: 351-353.

van der Gun, B.T.F., Wasserkort, R., Monami, M., Jeltsch, A., Raskó, T., Ślaska-Kiss, K., Cortese, R., Rots, M.G., de Leij, L.F.M.H., Ruiters, M.H.J., Kiss, A., Weinhold, E. and McLaughlin, P.M.J. (2008). Persistent down-regulation of the pancarcinoma-associated Epithelial Cell Adhesion Molecule via active intranuclear methylation. Int. J. Cancer 123: 484-489.

Kiss, A., Balikó, G., Csorba, A., Chuluunbaatar, T., Medzihradszky, K.F. and Alföldi, L. (2008). Cloning and characterization of the DNA region responsible for megacin A-216 production in Bacillus megaterium 216. J. Bacteriol. 190: 6448-6457.

Tímár, E., Venetianer, P. and Kiss, A. (2008). In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants. J. Bacteriol. 190: 8003-8008.

Darii, M.V., Cherepanova, N. A., Subach, O. M., Kirsanova, O. V., Raskó, T., Ślaska-Kiss, K., Kiss, A., Deville-Bonne, D., Reboud-Ravaux, M. and Gromova, E. S. (2009) Mutational analysis of the CG recognizing DNA methyltransferase SssI: Insight into enzyme-DNA interactions. Biochim. Biophys. Acta 1794: 1654-1662.

van der Gun, B. T. F., Maluszynska-Hoffman, M., Kiss, A., Arendzen, A., Ruiters, M. H. J., McLaughlin, P. M. J., Weinhold, E.; Rots, M. G. (2010) Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to downregulate the Epithelial Cell Adhesion Molecule. Bioconjugate Chem. 21: 1239-1245.

Raskó, T., Dér, A., Klement, É., Ślaska-Kiss, K., Pósfai, E., Medzihradszky, K. F., Marshak, D. R., Roberts, R. J. and Kiss, A. (2010) BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism. Nucleic Acids. Res. 38: 7155-7166.

Stier, I. and Kiss, A. (2010) The Type II restriction endonuclease MvaI has dual specificity. Nucleic Acids. Res. 38: 8231-8238.

Ślaska-Kiss, K., Tímár, E. and Kiss, A. (2012) Complementation between inactive fragments of SssI DNA methyltransferase. BMC Molecular Biology 13: 17. doi:10.1186/1471-2199-13-17