Nucleic Acids The expansion of structural databases for nucleic acids, proteins and nucleo-protein complexes, as well as continued progress in elucidating the sequence-dependent structural properties of DNA and RNA, will improve the accuracy with which large nucleo-protein complexes can be characterized. X-ray and NMR data will be used for better understanding the sequence dependence of different forms of DNA and also binding motifs of proteins in the major and minor grooves. Based on the results of computer simulations and high-resolution crystal structures, we have elucidated the extents of DNA sequence-dependent bending, twisting and stretching deformability. With these knowledge-based elasticity functions for DNA, we built a stereochemically feasible model for the tetrameric p53-DNA complex. The predicted directionality and magnitude of the DNA bending and twisting were subsequently confirmed by gel electrophoresis experiments. Now we are entering the second phase of this study, when the precise localization of the DNA bends is to be determined, and the complexes are to be compared for various p53 mutants. For this aim, we are using an original method, iodine-125 radioprobing, recently developed at NIH. Additional subjects include: (1)Gal Repressosome. Genetic data were used to determine the overall orientation of the gal repressors in a tetrameric structure. Knowledge-based computer modeling was employed to determine the trajectory of the DNA loop. According to our data, a similar (antiparallel) DNA loop is formed upon binding of the lac repressors to DNA. These findings imply that the antiparallel DNA looping may be a general feature of the condensed bacterial nucleoid, as opposed to the parallel DNA 'wrapping' around histones in eukaryotic chromatin. (2) DNA binding to mutant hSRY protein (human testis determining factor). When mutant hSRY binds to cognate DNA, the degree of DNA bending differs from the wild type case. Based on the knowledge of the sequence-dependent properties of DNA, we will analyze how the atomic interactions at the DNA-protein interface lead to an increase or decrease in the level of the DNA deformation in the complex, which, in turn, is involved in regulation of transcription. (3) Protein-Nucleic Acid Interactions. This is a genome analysis of protein binding sites for p53 sites in the human genome with a subsequent characterization of the conformational features of the adjacent DNA. Location of tandem p53 sites (putative storage pools), particularly on human chromosomes 21 and 22. (4) Iodine-125 Radioprobing - a novel method for tracing the DNA and RNA trajectories in nucleoprotein complexes. Radioprobing has been successfully applied to the CRP-DNA complex and to the T7 transcription elongation complex. In the latter case we were able to locate for the first time the DNA-RNA heteroduplex outside of the polymerase. This finding opens exciting prospects for the field of transcription-coupled mismatch repair. (5) Tetrameric p53-DNA complex. Iodine-125 radioprobing data indicate that upon binding to p53, DNA is severely bent and overtwisted at the CATG sequences, consistent with our computations and gel electrophoresis results. Thus, the apparent role of the sequence-specific DNA deformation is to control the p53-p53 interactions in order to form an interface on the surface of the p53 tetramer, suitable for the binding of other proteins, such as Mdm2. The next step is to analyze the effects of mutations at the p53 binding site, and of the p53 mutants, on stabilization of the tetrameric p53-DNA assemblage. (6) p53 and DNA helicases. Building models of their interactions and understanding how phophorylation controls interactions. Z01 BC 08371-17
