Reduction of a conserved Cys is essential for Myb DNA-binding.
AUTOR(ES)
Guehmann, S
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=312342Documentos Relacionados
- Elk-1 protein domains required for direct and SRF-assisted DNA-binding.
- The highly conserved DNA-binding domains of A-, B- and c-Myb differ with respect to DNA-binding, phosphorylation and redox properties
- REB1, a yeast DNA-binding protein with many targets, is essential for growth and bears some resemblance to the oncogene myb.
- Reduction in DNA-binding affinity of Cys2His2 zinc finger proteins by linker phosphorylation
- Drosophila and vertebrate myb proteins share two conserved regions, one of which functions as a DNA-binding domain.