Determination of Kd and relative Kd

The gel shift assay (Fried and Crothers 1981) was used to determine the K_d of Mif2 bound to the Cse4/CEN3 nucleosome. The equation used to calculate the K_d of nucleosome A relative to nucleosome B [relative K_d = (B_A/F_A)/(B_B/F_B)] was adopted from Liu-Johnson et al. (1986). Nucleosomes were reconstituted on DNA fragments labeled with either Alexa fluor 555 or Alexa fluor 647. To calculate a relative K_d, two different color-labeled nucleosomes or free DNA fragments (A and B) were mixed in the same binding reaction with increasing concentrations of purified Mif2c dimer or its derivatives. The reaction mixtures were analyzed by EMSA, and bands representing Mif2-bound (B) and free (F) species were scanned and quantified for fluorescence intensity.