Construction of an artificial tandem protein of the c-Myb DNA-binding domain and analysis of its DNA binding specificity

An artificial tandem protein was generated using the third repeat of the c- Myb DNA-binding domain, and its DNA binding affinity and specificity were a nalyzed by a filter binding assay, isothermal titration calorimetry, and su rface plasmon resonance. Although this artificial protein had the proper se condary structure, which is similar to the third repeat by itself, it could not bind to the expected base sequences specifically. Compared with the su ccessful results of the zinc finger fusion proteins with novel sequence spe cificities, the cooperativity between the adjacent repeats, observed in the c-Myb-DNA complex, should also be required for the DNA recognition by the artificial tandem protein. Using the previous analyses of the DNA binding s pecificities by Myb homologous proteins, the differences in the DNA recogni tion mechanisms between the animal and plant Myb domains are also discussed . (C) 1999 Academic Press.