Structure, mechanism and function of bacterial DNA methyltransferases
Zusammenfassung der Projektergebnisse
This project focused on the investigation of the mechanism of DNA recognition by formation of base specific contacts and by sequence dependent deformation of the DNA structure. The family of DNA- (adenine N6)-methyltransferases is a very interesting model system to study and understand the evolution of sequence specific DNA interaction, because it contains closely related enzymes which recognize different DNA sequences. For T4Dam, we showed that the DNA recognition is modular mediated by residues in a β-hairpin structure. In double mutant cycle experiments by combining enzyme mutants and methylation of modified target sites we showed that an exchange of Individual amino acid residues which interact with the DNA led to the loss of the ability to identify the corresponding base pairs in the DNA sequence. This approach was novel at that time and very powerful to unravel subtle functional consequences of sterical proximities observed in structural analysis. Interestingly, we could show that DNA recognition is altered in EcoDam despite the fact that EcoDam and T4Dam appear quite related and both recognize the same GATC sequence. Later we were able to reconstruct an evolutionary intermediate of the transition from a T4Dam to an EcoDam like DNA recognition. In a similar study with the related M.EcoRV strong evidence was provided for a central involvement of DNA bending and indirect readout in the DNA recognition of this enzyme. Moreover, we could show that M.EcoRV recognized its GATATC target site as an expanded GATC site. One important property of DNA methyltransferases is their processivity. We found previously that EcoDam is highly processive and interestingly a similar result was published for CcrM (Berdis et al., 1998). Therefore, we were interested to follow up on this finding and compare the mechanism of these two enzymes in more details. Surprisingly, it turned out that CcrM did not show processivity and the initial claim of this properties was based on a simple technical mistake in the setup of the experiment conducted by Berdis and colleagues. This finding was important for the field, since CcrM is known to methylate the genomic DNA in Caulobacter only at the end of the cell cycle after almost finishing of DNA replication. Since at this time DNA binding proteins will already be bound to the DNA and CcrM does not possess any motor domain, it was very difficult to imagine how this enzyme would slide along the DNA and methylate it in a processive reaction. In addition, we also studied the DNA recognition mechanism of the CcrM methyltransferase (GANTC) and found that DNA recognition was cooperative and it involves conformational changes and indirect readout. Later, we showed that an additional conserved C-terminal domain of CcrM actually is the DNA recognition part, indicated that CcrM is the first member of the delta subgroup of adenine-N6 methyltransferases. This result highlights the ongoing processes of domain shuffling and circular permutations in the molecular evolution of DNA methyltransferases. In summary, this project increased our understanding of the mechanisms by which DNA interacting proteins specifically recognize DNA by direct and indirect readout. These processes are essential in the regulation of the access of proteins to the genomic information, for example by transcription factors and sequence specific DNA recognition is the key to the cell type specific control of gene expression. In addition, we have explored pathways how DNA interacting proteins diverge through evolution and identified different processes leading to the change of DNA binding specificities. Currently we aim to apply prokaryotic adenine DNA methyltransferases to establish artificial epigenetic circuits in bacteria, which can process incoming signals and provide memory function. Such artificial epigenetic circuits could be useful in the toolbox of synthetic biology for example for bacterial live biosensors detecting industrial pollution.
Projektbezogene Publikationen (Auswahl)
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Transition from nonspecific to specific DNA interactions along the substrate-recognition pathway of dam methyltransferase. Cell. 2005 May 6;121(3):349-61
Horton JR, Liebert K, Hattman S, Jeltsch A, Cheng X
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Structure and substrate recognition of the Escherichia coll DNA adenine methyltransferase. J Mol Biol. 2006 Apr 28;358(2):559-70
Horton JR, Liebert K, Bekes M, Jeltsch A, Cheng X
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The M.EcoRV DNA-(adenine N6)- methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site. J Biol Chem. 2007 Dec 21;282(51):36942-52
Jurkowski TP, Anspach N, Kullshova L, Nellen W, Jeltsch A
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Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle. J Biol Chem. 2007 Aug 3;282(31):22848-55
Liebert K, Horton JR, Chahar S, Orwick M, Cheng X, jeltsch A
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Transition from EcoDam to T4Dam DNA recognition mechanism without loss of activity and specificity. Chembiochem. 2009 Oct 12;10{15):2488-93
Elsawy H, Podobinschi S, Chahar S, Jeltsch A
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Changing the DNA recognition specificity of the EcoDam DNA-(adenine-N6)-methyltransferase by directed evolution. J Mol Biol. 2010 Jan 8;395(1):79-88
Chahar S, Elsawy H, Ragozin S, Jeltsch A
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DNA interaction of the CcrM DNA methyltransferase: a mutational and modeling study. Chembiochem. 2012 Jun 18;13(9):1304-11
Albu RF, Zacharias M, Jurkowski TP, Jeltsch A
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The Caulobacter crescentus DNA-(adenine-N6)- methyltransferase CcrM methylates DNA in a distributive manner. Nucleic Acids Res. 2012 Feb;40(4):1708-16
Albu RF, Jurkowski TP, Jeltsch A
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Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding. Biophys Chem. 2012 Jan;160(1):28- 34
Bonnist EY, Liebert K, Dryden DT, Jeltsch A, Jones AC
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Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM. Biochimie, Volume 119, December 2015, Pages 60-67
Maier JAH, Albu RF, Jurkowski TP, Jeltsch A
