Family-wide Characterization of Methylated DNA Binding Ability of Arabidopsis MBDs |
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| Affiliation: | 1. School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 3216 Geelong, Australia;2. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, 3168 Melbourne, Australia;3. Department of Biochemistry and Pharmacology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3052 Melbourne, Australia;4. Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria 3002, Australia;5. Ophthalmology, University of Melbourne, Department of Surgery Melbourne, Victoria 3000, Australia;6. Department of Molecular and Translational Science, Monash University, 3168 Melbourne, Australia;1. School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK;2. Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK;3. Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore;4. Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK;5. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA;6. Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore;1. Department of Chemistry, University of Waterloo, Waterloo N2L 3G1, Ontario, Canada;2. Bristol Myers Squibb, Cambridge, MA 02140, United States;1. Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8021, Japan;2. Department of Bioscience, Faculty of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8021, Japan;3. Course of Biological Science, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8021, Japan;1. Dept. of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, United States;2. Center for Pathogen Genomics, National Institute of Infectious Diseases, Tokyo, Japan;3. Dept. of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus, OH, United States |
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| Abstract: | 13 MBD-containing genes (AtMBD1-13) have been identified in Arabidopsis thaliana so far, however, their DNA binding ability is still controversial. Here, we systematically measured the DNA binding affinities of these MBDs by ITC and EMSA binding assays, except for those of pseudogenes AtMBD3 and AtMBD13, and found that only AtMBD6 and AtMBD7 function as methylated DNA readers. We also found that the MBD of AtMBD5 exhibits very weak binding to methylated DNA compared to that of AtMBD6. To further investigate the structural basis of AtMBDs in binding to methylated DNA, we determined the complex structure of the AtMBD6 MBD with a 12mer mCG DNA and the apo structure of the AtMBD5 MBD. Structural analysis coupled with mutagenesis studies indicated that, in addition to the conserved arginine fingers contributing to the DNA binding specificity, the residues located in the loop1 and α1 are also essential for the methylated DNA binding of these MBDs in Arabidopsis thaliana, which explains why AtMBD5 MBD and the other AtMBDs display very weak or no binding to methylated DNA. Thus, our study here systematically demonstrates the DNA binding ability of the MBDs in Arabidopsis thaliana, which also provides a general guideline in understanding the DNA binding ability of the MBDs in other plants as a whole. |
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| Keywords: | DNA cytosine methylation MBD 5-methylcytosine DNA binding X-ray crystallography AtMBD" },{" #name" :" keyword" ," $" :{" id" :" k0035" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" __text__" ," _" :" Methyl-CpG-binding domain protein of " },{" #name" :" italic" ," _" :" Arabidopsis thaliana EMSA" },{" #name" :" keyword" ," $" :{" id" :" k0045" }," $$" :[{" #name" :" text" ," _" :" Electrophoretic mobility shift assay BiFC" },{" #name" :" keyword" ," $" :{" id" :" k0055" }," $$" :[{" #name" :" text" ," _" :" Bimolecular fluorescence complementation DDM1" },{" #name" :" keyword" ," $" :{" id" :" k0065" }," $$" :[{" #name" :" text" ," _" :" Decrease in DNA methylation 1 AtNTF2" },{" #name" :" keyword" ," $" :{" id" :" k0075" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" __text__" ," _" :" Nuclear transport factor 2 of " },{" #name" :" italic" ," _" :" Arabidopsis thaliana AtAGO4" },{" #name" :" keyword" ," $" :{" id" :" k0085" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" Arabidopsis thaliana" },{" #name" :" __text__" ," _" :" Argonoute 4 IDM" },{" #name" :" keyword" ," $" :{" id" :" k0095" }," $$" :[{" #name" :" text" ," _" :" Increased DNA Methylation |
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