@phdthesis{Elsawy2009,
  author    = {Elsawy, Hany Amin},
  title     = {Use of protein engineering to study DNA recognition by the E.coli DNA methyltransferase (EcoDam)},
  url       = {https://nbn-resolving.org/urn:nbn:de:101:1-201305237403},
  school    = {IRC-Library, Information Resource Center der Jacobs University Bremen},
  year      = {2009},
  abstract  = {DNA methylation carried out by DNA methyltransferases (MTases) is widespread in prokaryotes and eukaryotes. In this study, we have changed the target specificity of EcoDam from GATC to GATT by directed evolution, combining different random mutagenesis methods with restriction protection at GATT sites for selection and screening. The new methyltransferases are fully functional in vivo and in vitro. The EcoDam and T4Dam DNA-(adenine N6)-methyltransferases both methylate the adenine residue in GATC sites. They are highly related in amino acid sequence and structure but deviate in their contact to the first base pair of the target sequence. We have transplanted the T4Dam DNA recognition module into EcoDam and show that the EcoDam K9A/Y138R double mutant is highly active and specific. The evolutionary intermediates of this transition were studied as well: the EcoDam K9A variant showed low activity and loss of recognition of the first base pair, whereas; the EcoDam Y138R variant is fully active and specific. This result indicates that there exists a smooth evolutionary pathway changing the EcoDam DNA recognition mode to the T4Dam mode without loss of activity. Finally, in an attempt to enhance the preference of the L122A EcoDam for hemimethylated DNA to generate an artificial epigenetic system that can propagate patterns of GATC site methylation potentially useful for gene regulation, the L122, P134 and V133 residues were replaced with other amino acids using site directed mutagenesis. Our results showed that, in addition to L122A, the L122A/V133L EcoDam variant was able to sense the methylation status of the GATC target recognition site and methylated only hemimethylated DNA.},
  language  = {en}
}