Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila.
DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.
Choi
Tae-Yeong
TY
Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, Korea.
Park
So Young
SY
Kang
Ho-Sung
HS
Cheong
Jae-Hun
JH
Kim
Han-Do
HD
Lee
Bok Luel
BL
Hirose
Fumiko
F
Yamaguchi
Masamitsu
M
Yoo
Mi-Ae
MA
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D000477:Alkylating Agents; D004268:DNA-Binding Proteins; C099494:Dref protein, Drosophila; D029721:Drosophila Proteins; D016877:Oxidants; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D004247:DNA; D003545:Cysteine