DNA DEFORMATIONS AND INTERACTIONS IN COMPLEXES WITH PROTEINS

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The ultimate goals are: (1) to deduce the rules governing the sequence-dependent mechanical properties of the DNA duplex in complexes with proteins, (2) to apply these rules to specific cases of fundamental and practical interest. These goals can be achieved only through direct comparison between theory and experiment. Earlier, we developed a new approach to elucidate the DNA bending and twisting elasticities from the DNA variability in crystals. To complete these studies, in 1998-99 we analyzed the longitudinal deformations in DNA -- its stretching and compression. This kind of DNA motion is important for recombination, where the DNA extension serves more efficient recognition of the sister chromosomes, and is also advantageous for strand exchange. Our calculations provided the first detailed stereochemical model for the cooperative DNA stretching observed recently in micromanipulation experiments with single DNA molecules. In particular, we showed that extreme DNA stretching is accompanied by the B-A inter-family transition, similar to that observed for the complex with RecA protein. Importantly, we demonstrated that longitudinal deformations in DNA occur also in the complexes with TBP (TATA box binding proteins). This opens the possibilities for applying this kind of mechanistic model to other nucleoprotein systems, such as HIV reverse transcriptase and RNA polymerase bound to DNA and RNA strands. This work is in progress.Our theoretical analysis of the DNA complex with tumor suppressor protein p53 led to the gel electrophoresis experiments in which the predicted directionality and magnitude of DNA bending were confirmed (in collaboration with Dr. E. Appella, NCI). 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. To this aim, we are using an original method, iodine-125 radioprobing, developed recently at NIH. This approach has allowed us to detect the localized kinks in the DNA- CRP (CAP) complex in solution (in collaboration with Dr. R. Neumann, Clinical Center). Based on our calculations, we designed the necessary sequences, which will be used in the radioprobing experiments to visualize the local deformations in the p53-bound DNA.New subjects include:(i) DNA looping and transcription regulation; analysis of the multimeric complex with GalR repressor and HU protein (in collaboration with Dr. S. Adhya, NCI). We will apply our mechanistic rules for DNA to the mini-plasmid containing several hundred base pairs, in order to interpret the available data on the GalR-HU binding to DNA, and to suggest new experiments, that could elucidate the complicated 3D structure of this transcription regulation complex.(ii) DNA binding to mutant hSRY protein (human testis determining factor; in collaboration with Dr. M. Clore, NIDDK). When mutant hSRY binds to cognate DNA, the degree of DNA bending differs from the wild type case. Based on 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.Z01 BC 08371-16 - databases, DNA binding protein, DNA folding, Gene regulation, molecular models, transcriptional control, p, Tumor Suppressor,