Abstract
DNA methylation catalyzed by DNA methyltransferases (MTases) is widespread in prokaryotes. In an attempt to find EcoDam variants with enhanced preference for hemimethylated DNA, the L122, P134, and V133 residues were replaced with other amino acids using site directed mutagenesis, and the catalytic activity of all variants on unmethylated and hemimethylated substrates was studied. Our results showed that, in addition to L122A, the L122S and L122A/V133L EcoDam variants were able to sense the methylation status of the 5′-GATC-3′ double-stranded target recognition site and methylated only hemimethylated DNA.
Similar content being viewed by others
References
Jeltsch, A. (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases, ChemBioChem., 3, 274–293.
Jeltsch, A., Jurkowska, R. Z., Jurkowski, T. P., Liebert, K., Rathert, P., and Schlickenrieder, M. (2007) Application of DNA methyltransferases in targeted DNA methylation, Appl. Microbiol. Biotechnol., 75, 1233–1240.
Lobner-Olesen, A., Skovgaard, O., and Marinus, M. G. (2005) Dam methylation: coordinating cellular processes, Curr. Opin. Microbiol., 8, 154–160.
Joseph, N., Duppatla, V., and Rao, D. N. (2006) Prokaryotic DNA mismatch repair, Prog. Nucleic Acids Res. Mol. Biol., 81, 1–49.
Mahan, M. J., Heithoff, D. M., Sinsheimer, R. L., and Low, D. A. (2000) Assessment of bacterial pathogenesis by analysis of gene expression in the host, Ann. Rev. Genet., 34, 139–164.
Mahan, M. J., and Low, D. A. (2001) DNA methylation regulates bacterial gene expression and virulence, ASM News, 67, 356–361.
Horton, J. R., Liebert, K., Bekes, M., Jeltsch, A., and Cheng, X. (2006) Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase, J. Mol. Biol., 358, 559–570.
Jeltsch, A. (2008) Reading and writing of DNA methylation, Nat. Struct. Mol. Biol., 15, 1003–1004.
Horton, J. R., Liebert, K., Hattman, S., Jeltsch, A., and Cheng, X. (2005) Transition from non-specific to specific DNA interaction along the DNA recognition pathway of Dam methyltransferase, Cell, 121, 349–361.
Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein, Protein Sci., 4, 2411–2423.
Elsawy, H., Podobinschi, S., Chahar, S., and Jeltsch, A. (2009) Transition from EcoDam to T4Dam DNA recognition mechanism without loss of activity and specificity, ChemBioChem, 10, 2488–2493.
Roth, M., and Jeltsch, A. (2001) Biotin-avidin microtiter plate assay for quantitative analysis of DNA methylation by DNA methyltransferases, Nucleic Acids Res., 29, 3137–3144.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published in Russian in Biokhimiya, 2014, Vol. 79, No. 11, pp. 1548–1553.
Rights and permissions
About this article
Cite this article
Elsawy, H., Chahar, S. Increasing DNA substrate specificity of the EcoDam DNA-(adenine N6)-methyltransferase by site-directed mutagenesis. Biochemistry Moscow 79, 1262–1266 (2014). https://doi.org/10.1134/S0006297914110145
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297914110145