This research is directed toward the goal of understanding the structural organization of genomic DNA and associated nuclear proteins through detailed three-dimensional analysis of structures such as the nucleosome core particle, a small gene complete with promoter sequence, and more complex structures like the chromatosome and the polynucleosomal 30 nanometer fiber. The primary objective is to determine a medium resolution crystallographic structure of reconstituted nucleosome core particles containing a defined- sequence DNA. Nucleosomes reconstituted from purified chicken erythrocyte histones and a defined-sequence DNA have been crystallized. The structure will be solved to a resolution of approximately 3 Angstroms using data collected at a synchrotron light source. A combination of heavy-atom derivative multiple-wavelength anomalous diffraction and molecular replacement will be used to phase the X-ray diffraction data. The defined-sequence DNA of choice, constructed from """"""""one-half"""""""" of a 145 base-pair human alpha-satellite sequence, is a 146 base pair palindrome which confers overall two-fold symmetry to the nucleosome core particle. The palindrome-containing, two-fold symmetric nucleosomes display the highest resolution diffraction of any nucleosome crystals. Another defined-sequence DNA consists of an entire tRNA-Lys gene with the polymerase III promoter site located near the center of the nucleosome as in vivo. The entire gene and its internal promoter region will be observed in this structure. Other defined-sequence DNAs which form precisely phased nucleosomes for potential structure determination are a human alpha- satellite repeat and a sequence from SV40 flanking the nucleosome free region. This research program will eventually provide the near-atomic resolution information which is of fundamental importance to understanding the structures of DNA and chromatin as well as the controlling role of nucleosomes in modulating gene expression, retroviral integration target site selection, and DNA replication. The structural information attained will contribute to an understanding of the molecular basis of carcinogenesis, and should also be of interest to the Human Genome Project.
| Hanson, B Leif; Bunick, Gerard J (2007) Annealing macromolecular crystals. Methods Mol Biol 364:31-42 |
| Katz, Amy K; Li, Xinmin; Carrell, H L et al. (2006) Locating active-site hydrogen atoms in D-xylose isomerase: time-of-flight neutron diffraction. Proc Natl Acad Sci U S A 103:8342-7 |
| Hanson, B Leif; Alexander, Chad; Harp, Joel M et al. (2004) Preparation and crystallization of nucleosome core particle. Methods Enzymol 375:44-62 |
| Hanson, B Leif; Langan, Paul; Katz, Amy K et al. (2004) A preliminary time-of-flight neutron diffraction study of Streptomyces rubiginosus D-xylose isomerase. Acta Crystallogr D Biol Crystallogr 60:241-9 |
| Bunick, Christopher G; Nelson, Melanie R; Mangahas, Sheryll et al. (2004) Designing sequence to control protein function in an EF-hand protein. J Am Chem Soc 126:5990-8 |
| Hanson, B Leif; Schall, Constance A; Bunick, Gerard J (2003) New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium. J Struct Biol 142:77-87 |
| Hanson, B Leif; Harp, Joel M; Bunick, Gerard J (2003) The well-tempered protein crystal: annealing macromolecular crystals. Methods Enzymol 368:217-35 |
| Harp, J M; Hanson, B L; Timm, D E et al. (2000) Asymmetries in the nucleosome core particle at 2.5 A resolution. Acta Crystallogr D Biol Crystallogr 56:1513-34 |
| Timm, D E; Mueller, H A; Bhanumoorthy, P et al. (1999) Crystal structure and mechanism of a carbon-carbon bond hydrolase. Structure 7:1023-33 |
| Harp, J M; Hanson, B L; Timm, D E et al. (1999) Macromolecular crystal annealing: evaluation of techniques and variables. Acta Crystallogr D Biol Crystallogr 55:1329-34 |
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