Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

Cbf1P is a Saccharomyces cerevisiae chromatin protein belonging to the basi c region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding pr oteins. Cbf1p binds to a conserved element in the 5'-flanking region of met hionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cere visiae centromeric DNA, We have determined the apparent equilibrium dissoci ation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation ass ays, Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. Ho wever, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range be tween 3.2 and 12 nM, In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes, We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protei n and a deletion variant containing only: the bHLHzip domain of Cbf1p, Our results suggest that centromeric DNA outside the consensus CDEI sequence an d interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.