EM PHYSICAL STUDIES OF DNA BINDING PROTEIN COMPLEXES

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High resolution electron microscopy is used in physical studies of protein-nucleic acid complexes in two new areas. (1) The first is a comparison of complexes formed by the analogous but physically dissimilar DNA binding proteins of the filamentous viruses fd and Pfl; the fd gene 5 protein is one of the most intensively studied "model" DNA binding proteins presently available, and the Pfl analogue was recently discovered. How do these dissimilar proteins perform similar functions? The detailed three-dimensional structure of these helical complexes will be investigated, including conformational shifts, topological rearrangements, dissociation and reassembly, and a possible disk-to-helix transition accompanying a shift from an n=3 to an n=4 binding mode. Models will be developed for the arrangement of proteins and DNA in these complexes and for protein-DNA interactions as they relate to biological function. (2) The second area of study involves the use of the fd gene 5 protein as a "coating" on polynucleotides to amplify and directly visualize polynucleotide helical structure, an approach which preliminary investigations indicate should be feasible. This may provide a means for direct confirmation of unusual structures such as the left-handed "Z" or "loopout" forms of DNA, and for correlation with spectral data indicating the existence of these DNA forms in solution. The methodology for this work primarily involves the quantitative analysis of negatively-stained nucleoprotein structures visualized in an electron microscope at very high magnifications, using tilting/rotation in a goniometer stage to derive three-dimensional structural information. Circular dichroism (CD) spectroscopy is used for protein binding titrations and to correlate solution structural data (e.g., Z-DNA spectrum) with structures analyzed by microscopy. This work is health-related in that it is (1) a fundamental study of one of the best available examples of a class of proteins (helix-destabilizing proteins) that are likely to be universally involved in the processes of DNA replication, recombination, and repair, and (2) a study of unusual DNA structures that may enter into, for example, transcriptional control. Knowledge of these elemental biological processes is essential to understanding cellular function in health and disease.