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  Reduction of a conserved Cys is essential for Myb DNA-binding.

Gühmann, S., Vorbrüggen, G., Kalkbrenner, F., & Mölling, K. (1992). Reduction of a conserved Cys is essential for Myb DNA-binding. Nucleic Acids Research, 20(9), 2279-2286. doi:10.1093/nar/20.9.2279.

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Gühmann, S., Author
Vorbrüggen, G.1, Author              
Kalkbrenner, F., Author
Mölling, K., Author
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1Research Group of Molecular Cell Dynamics, MPI for biophysical chemistry, Max Planck Society, ou_578593              

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 Abstract: 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.

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Language(s): eng - English
 Dates: 1992-05-11
 Publication Status: Published in print
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 Rev. Type: Peer
 Identifiers: DOI: 10.1093/nar/20.9.2279
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Title: Nucleic Acids Research
Source Genre: Journal
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Pages: - Volume / Issue: 20 (9) Sequence Number: - Start / End Page: 2279 - 2286 Identifier: -