Identification of a conserved oxidation-sensitive cysteine residue in the NFI family of DNA-binding proteins.
The nuclear factor I (NFI) family of site-specific DNA-binding proteins plays a role in both transcription and adenovirus DNA replication. The DNA binding domain of NFI family members contains 4 cysteine residues (Cys-2, Cys-3, Cys-4, and Cys-5) that are conserved in all NFI proteins. Mutation of the Cys-2, Cys-4, and Cys-5 residues in the human NFI-C protein to several other amino acids abolished DNA binding, while 8 of 10 mutations of the Cys-3 residue had little or no effect on binding. Wild-type NFI-C was inactivated by N-ethylmaleimide in vitro, while the active Cys-3 mutant proteins were resistant to N-ethylmaleimide. Treatment of wild-type NFI in vitro with the oxidizing agent diamide also inactivated DNA binding, and subsequent reduction with dithiothreitol restored binding activity. The active Cys-3 mutant NFI proteins were resistant to diamide-inactivation, indicating that the Cys-3 residue is required for modulation of DNA-binding by oxidation state. These studies indicate that oxidative-inactivation can play an important role in the modifying NFI-DNA-protein interactions. The presence of this nonessential Cys-3 residue in all known NFI proteins raises the possibility that it may function in a manner similar to redox-sensitive cysteine residues found in other site-specific DNA-binding proteins.
Bandyopadhyay
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Department of Cancer Biology, Cleveland Clinic Foundation Research Institute, Ohio 44195.
Gronostajski
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RM
D000595:Amino Acid Sequence; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010084:Oxidation-Reduction; D014157:Transcription Factors / Q000737:chemistry*; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; C497647:NFIC protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D003545:Cysteine; D005033:Ethylmaleimide