DNA ENZYME DATA COLLECTION

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Synchrotron radiation is essential for data collection from weakly diffracting crystals of macromolecular nucleic acids. Structural studies in this project focus on a DNA enzyme that cleaves RNA with high specificity. The combined DNA:RNA complex has been crystallized in the hexagonal space group P6(1)22, and the structure has been determined with the use of 5 heavy atom derivatives and refined at 3.0 E resolution. The structure of the 82-nucleotide DNA:RNA complex reveals that the DNA enzyme in the hexagonal crystals has rearragned to form a dimer mediated by a palidromic sequence in the conserved core of the enzyme. However, the structure provides an atomic resolution model for Holliday junctions, a 4-stranded structure required for genetic recombination. In order to capture an active conformation of the DNA enzyme a combinatorial crystallization screen has been carried out on 81 sequence variants using 3880 individual conditions. The screen has resulted in 40 new crystal forms. These crystal forms need to be evaluated for diffraction characteristics and the best crystal forms need to be employed for structure determinations. These studies will provide critical insight into the mechanism of the DNA enzyme. This mechanism is relevant to the enzyme function and is of fundamental interest with respect to the function of ribozymes and protein enzymes. The DNA enzyme is currently being used in clinical trials and so the results are also relevant to therapeutic applications.