title: Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins.
abstract: Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides.
authors.affiliation: Emory University School of Medicine, Atlanta, GA, United States.
authors.forename: A
authors.initials: A
authors.lastname: Patel
authors.affiliation: Emory University School of Medicine, Atlanta, GA, United States.
authors.forename: H
authors.initials: H
authors.lastname: Hashimoto
authors.affiliation: Emory University School of Medicine, Atlanta, GA, United States. Electronic address: xzhan02@emory.edu.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Emory University School of Medicine, Atlanta, GA, United States. Electronic address: xcheng@emory.edu.
authors.forename: X
authors.initials: X
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001483:Base Sequence; D000070580:CYS2-HIS2 Zinc Fingers; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D004247:DNA
keywords: C2H2 zinc finger; DNA methylation; DNA modification; Epigenetics; Ionic strength; Polyethylenimine