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 prote in, 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 Sou thwestern analyses confirm that a 70 kDa polypeptide interacts specifi cally with cruciform DNA. Microsequence analysis of tryptic peptides o f the 70 kDa CBP reveals that it is 100% homologous to the 14-3-3 fami ly of proteins and shows that CBP contains the epsilon, beta, gamma, a nd zeta isoforms of the 14-3-3 family, In addition to polypeptides wit h 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 epsilon isoform of 14-3-3. Cruciform-specifi c binding activity is also detected in 14-3-3 proteins purified from s heep brain. Immunofluorescene studies confirm the presence of the epsi lon, beta, and zeta 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 associ ated with the 14-3-3 family.