Deep Enzymology Studies on DNA Methyltransferases Reveal Novel Connections between Flanking Sequences and Enzyme Activity |
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| Institution: | 1. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel;2. Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot, Israel;3. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel;1. Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;2. Institute for Molecular Virology, University of Minnesota – Twin Cities, Minneapolis, MN 55455, USA;1. Max‐Delbrück‐Center for Molecular Medicine in the Helmholtz Association (MDC), Laboratory for Neuroproteomics, Berlin, Germany;2. Max‐Delbrück‐Center for Molecular Medicine in the Helmholtz Association (MDC), Laboratory for Mobile DNA, Berlin, Germany;1. Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel;2. Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel;3. Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, 69978 Tel-Aviv, Israel;1. Department of Biochemistry, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada;2. Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada;1. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA;2. Department of Chemistry, Grand Valley State University, Allendale, MI 49401, USA |
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| Abstract: | DNA interacting enzymes recognize their target sequences embedded in variable flanking sequence context. The influence of flanking sequences on enzymatic activities of DNA methyltransferases (DNMTs) can be systematically studied with “deep enzymology” approaches using pools of double-stranded DNA substrates, which contain target sites in random flanking sequence context. After incubation with DNMTs and bisulfite conversion, the methylation states and flanking sequences of individual DNA molecules are determined by NGS. Deep enzymology studies with different human and mouse DNMTs revealed strong influences of flanking sequences on their CpG and non-CpG methylation activity and the structures of DNMT-DNA complexes. Differences in flanking sequence preferences of DNMT3A and DNMT3B were shown to be related to the prominent role of DNMT3B in the methylation of human SATII repeat elements. Mutational studies in DNMT3B discovered alternative interaction networks between the enzyme and the DNA leading to a partial equalization of the effects of different flanking sequences. Structural studies in DNMT1 revealed striking correlations between enzymatic activities and flanking sequence dependent conformational changes upon DNA binding. Correlation of the biochemical data with cellular methylation patterns demonstrated that flanking sequence preferences are an important parameter that influences genomic DNA methylation patterns together with other mechanisms targeting DNMTs to genomic sites. |
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| Keywords: | DNA methyltransferase DNA recognition Enzyme mechanism DNA methylation |
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