Ultra-high resolution DNA crystallography

This thesis represents results of experiments involved in ultra-high resolution x-ray crystal structures of DNA. Detailed analysis of DNA coordinates of increased accuracy, refinement of individual anisotropic B-factors, visualization of disorder, counterions and hydration are presumably facilitated by the increased information of ultra-high resolution structures. However, recent survey of available DNA crystal structures with the data that extends beyond 1A has brought a few areas of considerations; local conformational flexibility of DNA and counterions and how they have been interpreted in previous studies. While the results given here demonstrate both structural and surrounding details of DNA that may be undetected at lower resolution, some of the technical aspects of interpreting ultra-high resolution data is also addressed.;First part describes the 0.96A crystal structure of one turn of G/C rich B-DNA. Providing much improved data, thorough analysis revealed details of B-DNA conformation, as well as greater information on the local variations that may occur within such helices. Many phosphate groups are shown to assume in alternate states, and such static disorders are extended to the base pairs at the center of the duplex. One phosphate group is shown to transition from B-helical subtype BI to BII. Ultrahigh resolution structure also enabled comprehensive analysis of localized divalent cations. Many are observed to be disordered and/or partially occupied. A strong correlation observed between these counterions and static disorders within helices is also described.;Locating monovalent cations is also examined in order to address fundamental questions about the interaction of B-DNA and monovalent cations, as well as to examine the issue of charge disparity between DNA and counterions through the large majority of published DNA crystal structures. The locations of counterions around B-DNA were determined through 0.98A and 0.87A resolution x-ray diffraction of crystals. These are the first examples of <1A resolution B-DNA crystal structures within which the monovalent cations were analyzed. The majority of monovalent cations are localized in the G-tract major groove; however a few are also identified in the minor groove at TpG steps. The mode of coordination of these monovalent cations to the base functional groups and backbone of B-DNA are in essence similar, yet are observed to differ from previously determined monovalent cation sites in different sequences of B-DNA. The differences observed in coordination suggest the sequence dependence of cation localization.;Lastly, a 1.1A resolution x-ray crystal structure of an intercalative complex between [d(CGATCG)]2 and proflavine is described. Two proflavine molecules are observed to intercalate at CpG steps at the both terminals of the DNA duplex. Proflavine orients itself so that its terminal amines are toward the major groove. Proflavine molecules are in close van der Waals contacts with the adjacent C/G and G/C base pairs, positioning themselves in the pockets created by these unwound steps. The mode of intercalation by proflavine molecules is observed to be largely through stacking interaction, however several water and ion mediated contacts to the neighboring bases and phosphodiester backbone are also observed. Helical parameters of the current structure reveal that intercalation causes DNA to unwind and lengthen dramatically at the intercalation sites. These changes in helical parameters are found to be similar to the published intercalative complex of DNA with another high planarity intercalator, ellipticine.