Human Cruciform Binding Protein Belongs to the 14-3-3 Family

Cruciform DNA has been implicated in the initiation of DNA replication. Recently, we identified and purified from human (HeLa) cells a protein, CBP, with binding specificity for cruciform DNA. We have reported previously that the CBP activity sediments at approximately 66 kDa in a glycerol gradient. Here, photochemical cross-linking studies and Southwestern analyses confirm that a 70 kDa polypeptide interacts specifically with cruciform DNA. Microsequence analysis of tryptic peptides of the 70 kDa CBP reveals that it is 100% homologous to the 14-3-3 family of proteins and shows that CBP contains the ε, β, γ, and ζ isoforms of the 14-3-3 family. In addition to polypeptides with the characteristic molecular mass of 14-3-3 proteins (30 and 33 kDa), CBP also contains a polypeptide of 35 kDa which is recognized by an antibody specific for the ε isoform of 14-3-3. Cruciform-specific binding activity is also detected in 14-3-3 proteins purified from sheep brain. Immunofluorescene studies confirm the presence of the ε, β, and ζ isoforms of 14-3-3 proteins in the nuclei of HeLa cells. The 14-3-3 family of proteins has been implicated in cell cycle control, and members of this family have been shown to interact with various signaling proteins. Cruciform binding is a new activity associated with the 14-3-3 family.