Site-specific modification of single cysteine Pax3 mutants reveals reciprocal regulation of DNA binding activity of the paired and homeo domain.
The mechanism by which the paired domain (PD) and the homeo domain (HD) act together in the intact Pax3 protein to recognize DNA is unclear and was studied in a Pax3 mutant (Pax3-CL) devoid of cysteines. Pax3-CL binds to PD (P6CON-P3OPT sites) and HD (P2, P1/2 sites) DNA site sequences with near wild-type activity but, contrary to Pax3, in a N-ethyl maleimide (NEM) insensitive fashion. The Pax3-CL backbone was used for cysteine scanning mutagenesis and for site-specific NEM modification. Five single cysteine replacements were independently introduced in the PD, while eight were inserted in the HD. NEM sensitivity of PD and HD DNA binding was investigated in DNA-binding competent mutants. In the PD mutant C82, NEM abrogated DNA binding by the PD but also abolished DNA binding by the Cys-less HD. Likewise, in the HD mutant V263C, NEM modification abrogated DNA binding not only by the HD, but also by the Cys-less PD. The transfer of NEM sensitivity to the PD seen in V263C was specific and not due to simple loss of HD DNA binding since alkylation of adjacent V265C and S268C, although impairing HD DNA binding did not affect PD DNA binding. Thus, the PD and HD do not function as independent DNA binding modules in Pax3 but seem functionally interdependent.(1)
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019556:COS Cells; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors
D001643:Bridged Bicyclo Compounds; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C068686:Pax3 protein, mouse; C037094:dibromobimane; D004247:DNA; D003545:Cysteine; D005033:Ethylmaleimide