title: Interplay of binding stoichiometry and recognition specificity for the interaction of MBD2b protein and methylated DNA revealed by affinity capillary electrophoresis coupled with laser-induced fluorescence analysis.
abstract: The methyl-CpG binding domain (MBD) family proteins can specifically bind methylated DNA sequences and thereby mediate gene transcription. In this study, we used neutral capillary electrophoresis coupled with laser-induced fluorescence to investigate the interactions of DNA and MBD2b, a model MBD family protein with the highest affinity. For this purpose, we synthesized 13 double-stranded oligonucleotides of varying length (20 bp to 80 bp) and of varying methylation density. The sequences of these oligonucleotides were adapted from a frequently methylated promoter region of human p16(INK4a) gene. We demonstrate that multiple MBD2b proteins can bind to one DNA molecule with a DNA length-dependent binding stoichiometry. Each MBD2b protein can occupy 20 nucleotides in a bound DNA molecule regardless of the methylation status of DNA. By binding multiple MBD2b proteins (up to four protein molecules) to one dsDNA molecule (80 bp), methylated and unmethylated DNA were bound at similar percentages. Although the total amount of the DNA-MBD2b complexes increases with increasing DNA length for both unmethylated and methylated DNA, the DNA-MBD2b complexes of 1:1 display more than 10-fold higher affinity for methylated DNA (e.g., 40 bp DNA) accompanying a 20-fold lower dissociation rate constant. Hence, our study clarifies for the first time that the specificity of MBD2b to methylated DNA decreases as more MBD2b monomers binding to the same region of DNA. Additionally, this study opens a new venue to improve MBD protein-based assays for detecting DNA methylation.
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences , Beijing 100085 China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Zou
authors.forename: Dapeng
authors.initials: D
authors.lastname: Zhang
authors.forename: Shengquan
authors.initials: S
authors.lastname: Liu
authors.forename: Bailin
authors.initials: B
authors.lastname: Zhao
authors.forename: Hailin
authors.initials: H
authors.lastname: Wang
mesh_terms: D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D007834:Lasers; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C115368:MBD2 protein; D004247:DNA