The central objective of this research program is to understand the structural basis of nucleic acid recognition in protein/DNA and drug/DNA complexes that function in gene regulation and chromosome organization. The focus is on identifying structural, dynamic and thermodynamic features diagnostic of DNA recognition in large biological assemblies. The proposed studies will exploit methods of polarized Raman, UV-resonance Raman and near-IR Raman spectroscopy and complementary techniques developed in the investigators' laboratory. The results will provide new information on DNA bending, supercoiling, structure transformation and thermodynamic stability essential to gene function.
The specific aims are to: (1) Determine local and global DNA conformations and specific sites of base and backbone interactions in complexes of DNA with gene regulatory proteins and drugs. (2) Introduce novel Raman-based approaches to characterize DNA bending, folding and supercoiling and to identify the molecular mechanisms underlying such structural transformations. (3) Develop new Raman approaches and improve existing methodologies for probing DNA structure, thermodynamics and recognition. The project will provide an experimental basis for probing details of DNA geometry at protein/DNA and drug/DNA interfaces and investigating DNA solution structures and stabilities in assemblies that are not amenable to study by alternative methods. Targeted for study are genomic sequences, gene regulatory factors and pharmacological agents for which complementary genetic and/or biochemical information is either available or obtainable. These include plasmids of known superhelical density, custom-designed DNA minicircles, HU and IHF architectural proteins, the high-mobility-group (HMG) box of the activator encoded by the sex-determining region of the human Y chromosome (hSRY), telomere-end-binding proteins and telomeric DNA of Oxytricha nova, and representative intercalating and minor-groove-binding drugs. We shall also investigate model DNA oligomers and polymers of defined sequence to assist spectral interpretations on native assemblies. These studies will build upon the foundation established during prior support periods and are expected to yield new insights into biologically important DNA structural phenomena. ? ?
| Nemecek, Daniel; Stepanek, Josef; Thomas Jr, George J (2013) Raman spectroscopy of proteins and nucleoproteins. Curr Protoc Protein Sci Chapter 17:Unit17.8 |
| Tsuboi, Masamichi; Tsunoda, Masaru; Overman, Stacy A et al. (2010) A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1. Biochemistry 49:1737-43 |
| Tsuboi, Masamichi; Benevides, James M; Thomas Jr, George J (2009) Raman tensors and their application in structural studies of biological systems. Proc Jpn Acad Ser B Phys Biol Sci 85:83-97 |
| Benevides, James M; Danahy, Jessica; Kawakami, Jessica et al. (2008) Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation. Biochemistry 47:3855-62 |
| Nemecek, Daniel; Gilcrease, Eddie B; Kang, Sebyung et al. (2007) Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22. J Mol Biol 374:817-36 |
| Benevides, J M; Li, T; Lu, X J et al. (2000) Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex. Biochemistry 39:548-56 |
