Nucleic Acids Research, 1992, Vol. 20, No. 9 2279-2286
© 1992
MOLECULAR BIOLOGY |
Reduction of a conserved Cys is essential for Myb DNA-binding
Max-Planck-Institut für Molekulare Genetik Abt. Schuster, Ihnestrasse 73, D-1000 Berlin 33, Germany
*To whom correspondence should be addressed
Received February 3, 1992. Revised March 31, 1992. Accepted March 31, 1992.
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 R1R3 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 supple mented 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 trans fected 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.
+Present address: Institut für Pharmakologie, Freie Universität, Thielallee 69-73, D-1000 Berlin 33, Germany
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