NMR STRUCTURAL STUDIES OF CBFA RUNT DOMAIN DNA COMPLEX

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Core binding factor (CBF) was originally identified as a DNA-binding protein that specifically binds to the asymmetric sequence PyGPyGGT, corresponding to the highly conserved "core" site in mammalian type C retrovirus enhancers. CBF binding sites have subsequently been identified in a number of T cell specific genes, providing evidence for the role of CBF as a T-cell transcription factor. Additional evidence for the importance of CBF has come from knockouts of both genes in mice which were shown to be embryonic lethal and which also were shown to have blockage in hematopoietic development. Isolation and subsequent cloning of CBF showed the protein to be a heteromer consisting of an alpha subunit and a beta subunit. The alpha subunit contacts the DNA directly, whereas the beta subunit does not, as indicated by the lack of any changes in the number of phosphate contacts made by alpha in the presence of beta. Binding of the beta subunit to the alpha subunit increases the affinity of the alpha subunit for DNA sixfold without altering the sequence specificity. The alpha subunit contains a 128 amino acid region displaying a high homology to the Drosophila segmentation protein called Runt. This domain is referred to as the Runt domain and has been shown to be responsible for the both the DNA and beta-binding capabilities of the alpha subunit. Two of the four genes encoding CBF subunits are proto-oncogenes commonly activated in human leukemias. The inversion and translocations identified in these genes are associated with 30 percent of de novo acute myeloid leukemias in humans. The importance of CBF in leukemia as well as in its normal role as a transcription factor makes elucidation of its function at the molecular level extremely interesting and potentially therapeutically useful. In addition, the lack of any resemblance of either subunit to any known structural motifs makes them important targets for structure determination. The overall objective of our work is the thorough biophysical and structural characterization of the functional domains of the two subunits of CBF as well as the relevant complexes involved. We are employing NMR spectroscopy for the structure determinations of CBFbeta(1-141) as well as a Runt domain-DNA complex. The aims of this proposal are to determine the structure of the Runt domain-DNA complex and map the CBFbeta binding site on the Runt domain.