Reduction of a conserved Cys is essential for Myb DNA-binding.
The human c-Myb gene product is a regulator of transcription with intrinsic DNA-binding activity located in two of three aminoterminal repeats R2R3. Three purified recombinant c-Myb proteins, a 42 kD protein corresponding to the amino-terminal half (HM42), and two proteins representing R2R3 or R1R2R3 (HMR23 and HMR123) have been analyzed either as purified proteins or present in bacterial extracts in gelshift analyses using a high-affinity DNA oligonucleotide. The purified proteins are inactive in DNA-binding unless supplemented with a reducing agent such as dithiothreitol (DTT) in vitro. Alternatively a cellular nuclear extract (Nex) from HeLa cells strongly activates the binding. This effect is dose-dependent and sensitive to heat. The Nex does not lead to changes in the Myb-DNA mobility shift assay excluding a direct association of the complex with a cellular component. Site-directed mutagenesis of the aminoacid residue 130, a single conserved cysteine in HMR23 to serine almost completely abolishes DNA binding. Oxidation by diamide or alkylation by N-ethylmaleimide (NEM) of the Myb-proteins in vitro inhibit their interaction with DNA whereby the diamide effect is reversible by addition of excess of DTT. Nex prepared from COS cells transfected with c-myb leads to Myb-DNA interaction which is not responsive to DTT but sensitive to NEM and diamide. Our data indicate that the reduced cysteine of Myb is essential for its DNA-binding and that Myb function may be regulated by a reduction-oxidation mechanism.
Guehmann
S
S
Max-Planck-Institut für Molekulare Genetik, Abt. Schuster, Berlin, Germany.
Vorbrueggen
G
G
Kalkbrenner
F
F
Moelling
K
K
D000478:Alkylation; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb
D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D003958:Diamide; D004247:DNA; D003545:Cysteine