STRUCTURAL CHARACTERIZATION OF THE NUCLEOSOME-CHD1 COMPLEX

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. CHD1 is an ATP-driven multi-domain chromatin-remodeling protein. While it is known that an ATPase domain drives the motion of DNA along the histone-core by breaking histone-DNA contacts, the DNA-binding domain also binds the flanking regions of the nucleosome. We and others have determined the high resolution structures of the individual components but the stoichiometry and exact orientation on the nucleosome as the remodeling cycle progresses is yet to be elucidated. We hope to obtain precise information about the spatial organization of the different domains of CHD1 with respect to the nucleosome by docking high-resolution structures for the individual components into SAXS envelopes for the whole complex. We will obtain SAXS data for several variants of the nucleosome-CHD1 complex, and their individual components. We have purified homogeneous samples of wild-type CHD1 and chimeric constructs containing a sequence specific DNA-binding domain from the E.coli protein AraC. We have also reconstituted nucleosomes with DNA containing a strategically placed recognition sequence for AraC. We have confirmed formation of a 1:2 nucleosome-CHD1 complex on a gel-filtration column and a native polyacrylamide gel. We have also used biochemical functional assay to show that the chimeric constructs are enzymatically competent. We have further characterized and confirmed the mono-disperse nature of the complexes we have purified using SEC-MALS (Size Exclusion Chromatography - Multiple Angle Light Scattering). We have also obtained preliminary SAXS data that corroborates the homogeneity of our samples.