Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding.
The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.
Walker
L J
LJ
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, United Kingdom.
Robson
C N
CN
Black
E
E
Gillespie
D
D
Hickson
I D
ID
D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C058452:HAP1 protein, S cerevisiae; C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D003545:Cysteine