Identification and Characterization of the DNA Binding Domain of CpG-binding Protein

Jeong Heon Lee, Kui Shin Voo, David G. Skalnik

Research output: Contribution to journalArticle

105 Citations (Scopus)

Abstract

CpG-binding protein is a transcriptional activator that exhibits a unique DNA binding specificity for unmethylated CpG motifs. CpG-binding protein contains a cysteine-rich CXXC domain that is conserved in DNA methyltransferase 1, methyl binding domain protein 1, and human trithorax. In vitro DNA binding assays reveal that CpG-binding protein contains a single DNA binding domain comprised of the CXXC domain and a short carboxyl extension. Specific mutation to alanine of individual conserved cysteine residues within the CXXC domain abolishes DNA binding activity. Denaturation/renaturation experiments in the presence of various metal cations demonstrate that the CXXC domain requires zinc for efficient DNA binding activity. Ligand selection of high affinity binding sites from a pool of degenerate oligonucleotides reveals that CpG-binding protein interacts with a variety of sequences that contains the CpG dinucleotide with a consensus binding site of (A/C)CpG(A/C). Mutation of the CpG motif(s) present within ligand-selected oligonucleotides ablates the interaction with CpG-binding protein, and mutation to thymine of the nucleotides flanking the CpG motifs reduces the affinity of CpG-binding protein. Hence, a CpG motif is necessary and sufficient to comprise a binding site for CpG-binding protein, although the immediate flanking sequence affects binding affinity.

Original languageEnglish (US)
Pages (from-to)44669-44676
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number48
DOIs
StatePublished - Nov 30 2001
Externally publishedYes

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Carrier Proteins
DNA
Binding Sites
Oligonucleotides
Cysteine
Thymine Nucleotides
Mutation
Ligands
Denaturation
Methyltransferases
Alanine
Cations
Zinc
Assays
Metals
Proteins
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification and Characterization of the DNA Binding Domain of CpG-binding Protein. / Lee, Jeong Heon; Voo, Kui Shin; Skalnik, David G.

In: Journal of Biological Chemistry, Vol. 276, No. 48, 30.11.2001, p. 44669-44676.

Research output: Contribution to journalArticle

Lee, Jeong Heon ; Voo, Kui Shin ; Skalnik, David G. / Identification and Characterization of the DNA Binding Domain of CpG-binding Protein. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 48. pp. 44669-44676.
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