SITE-DIRECTED MUTAGENESIS SUPPORTS A 3-DIMENSIONAL MODEL OF THE RUNT DOMAIN

The ''runt domain'' (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-b inding domain of a new family of transcription factors; the RD protein s. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental Processes, from sex det ermination and neurogenesis in Drosophila to hematopoiesis and osteobl ast differentiation in mouse and human. The RD confers DNA binding abi lity and mediates the interaction of mammalian RD proteins with the be ta-subunit (CBF beta), which enhances the DNA binding. The primary seq uence of RD shows no similarity to other known DNA-binding motifs and its thee-dimensional (3D) structure is not known. We employed molecula r modeling-based mutagenesis to generate a 3D model of RD. Fold recogn ition programs identified the palm subdomain of rat DNA polymerase bet a as the most likely fold for RD. In the predicted model, the RD regio n which interacts with DNA contains two arginine residues, R130 and R1 35, which appear to be in close contact with the major groove of the D NA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated t hat mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBF beta. The data provide important clues abo ut the possible 3D structure of the RD and its interaction with the co re DNA motif. (C) 1998 Academic Press Limited.