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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034293-01
Application #
3285009
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1985-01-01
Project End
1987-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Texas-Dallas
Department
Type
Schools of Arts and Sciences
DUNS #
City
Richardson
State
TX
Country
United States
Zip Code
75080
Gray, C W (2001) Electron microscopy of protein-nucleic acid complexes. Uniform spreading of flexible complexes, staining with a uniform thin layer of uranyl acetate, and determining helix handedness. Methods Mol Biol 148:579-87
Thompson, T M; Mark, B L; Gray, C W et al. (1998) Circular dichroism and electron microscopy of a core Y61F mutant of the F1 gene 5 single-stranded DNA-binding protein and theoretical analysis of CD spectra of four Tyr --> Phe substitutions. Biochemistry 37:7463-77
Gray, C W (1994) Visualization of unshadowed DNA by electron microscopy. Adsorption to a bacitracin film. Methods Mol Biol 22:13-23
Gray, C W (1994) Electron microscopy of protein-nucleic acid complexes. Enhanced high-resolution shadowing. Methods Mol Biol 22:1-12
Gray, C W (1994) Electron microscopy of protein-nucleic acid complexes. Uniform spreading and determination of helix handedness. Methods Mol Biol 30:347-56
Gray, C W; Slaughter, C A; DeMartino, G N (1994) PA28 activator protein forms regulatory caps on proteasome stacked rings. J Mol Biol 236:7-15
Dick, L R; Geraldes, C F; Sherry, A D et al. (1989) 13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate. Biochemistry 28:7896-904
Gray, C W (1989) Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA. J Mol Biol 208:57-64
Antao, V P; Gray, C W; Gray, D M et al. (1986) Circular dichroism of two conformations of poly[d(G-C)] induced by low pH. Nucleic Acids Res 14:10091-112