Structure-function, immunochemistry and in vivo detection of DNA quadruplexes.
Changes in chromosomal DNA length at the molecular ends - the telomeres - are correlated with changes in cellular aging and cancer. Guanine-containing single-stranded termini can form novel four-stranded structures which can inhibit the recognition of telomeric DNA by telomere terminal transferase, a key RNA-containing enzyme which helps ensure that the DNA functions properly. Telomerase recognition controls chromosomal structural changes that lead to the new cell cycle, thereby regulating cellular turnover (apoptosis) and aging. When this control is breached, the number of life cycles increases, resulting in propagation of potentially mutant or metastatic cells - and cancer. Our work on the structural diversity, energetics and dynamics of G-rich sequences leads us to suspect that four-stranded DNAs might play a key role in regulating cell cycle progression and limiting cancerous cell growth. We are characterizing properties of quadruplex DNAs that could change the functional status of the strands in terms of protection, transcription, replication, legitimate and illegitimate recombination and chromosomal turnover. These studies involve in vitro analysis using spectroscopic approaches to determine structures, energies and effector-dependent interconvertability, and in vivo immunochemical approaches to determine localization and functional characteristics.
Structure and mechanism of maize ribosome inactivating protein.
Maize seeds produce a 32 kilodalton protein which is activated by a specific proteolysis event to produce an enzyme which recognizes and removes a specific nucleotide residue from ribosomal RNA, thereby inactivating the protein synthesis apparatus. This "ribosome inactivating protein" - or RIP - presumably recognizes the "Achilles heel" on ribosomes of invading pests which might compete with the developing corn plant for the foodstore in the seed. By doing so, it is thought to mediate a natural form of pest control. Click the image on the left for a larger picture of RIP. This project involves using X-ray crystallography methods to determine the structure of the protein - before and after proteolytic activation - and determining factors which facilitate the activation and RNA-binding events. The long-range goal is to harness and tune this novel activity for biotechnological applications such as "magic bullet" strategies for disease control.
Selected research publications
(1) Quadruplex DNAsRoss, W. S. and Hardin, C. C. (1994) Ion-Indiced Stabilization of The G-DNA Quadruplex: Free Energy Perturbation Studies. J. Am. Chem. Soc. 116, 6070-6080. Allosteric Interactions Between DNA Strands and Monovalent Cations in DNA Quadruplex Assembly: Thermodynamic Evidence for Three Linked Association Pathways (1997) Hardin, C. C., Corregan, M. J., Lieberman, D. V. and Brown II, B. A. Biochemistry 36, 15428-15450. Isolation and Characterization of a monoclonal Anti-Quadruplex DNA Antibody From Autoimmune "Viable Motheaten" Mice (1998) Brown II, B. A., Li, Y., Brown, J. C., Hardin, C. C., Roberts, J. F., Pelsue, S. C. and Schultz, L. D. Biochemistry 37, 16325-16337. Construction and Characterization of a Quadruplex DNA Selective Single-Chain Autoantibody from a Viable Motheaten Mouse Hybridoma With Homology To Telomeric DNA Binding Proteins (1998) Brown, J. C., Brown II, B. A., Li, Y., Brown, J. C., Hardin, C. C. Biochemistry 1998 37, 16338-16348. Extraction of Information On The Buildup and Consumption of Reactive Intermediates From quadruplex DNA Assembly Time Courses (2004) Lieberman, D. V. and Hardin, C. C. Biochim. Biophys. Acta 1679, 59-64.
(2) Collaborative StudiesSpecific Interaction of Glyceraldehyde 3-Phosphate Dehydrogenase With The 5'-Nontranslated RNA of Hepatitus A Virus (1996) Schultz, D., Hardin, C. C. and Lemon, S. M. J. Biol. Chem. 271, 14134-14142. Zhang, G., Foegeding, E. A. and Hardin, C. C. (2004) Effect of Sulfated Polysaccharides On Heat-Induced Structural Changes in Β-Lactoglobulin, J. Ag. and Food. Chem. 52, 3975-3981.
Patent:Antibodies That Selectively Bind Quadruplex Nucleic Acids, United States Patent No. 6,001,657, Issued Dec. 14, 1999 , Hardin, C. C., Brown II, B. A., Roberts, J. F., Pelsue, S. C.
Review Articles:Nucleic Acids Structural Energetics (2000) Hardin, C. C. and Moon, A. F., in Encyclopedia of Analytical Chemistry: Applications Theory and Instrumentation. pp. 5072-5105 (Meyers, R. A., Ed.) John Wiley and Sons. Thermodynamic and Kinetic Characterization of the Dissociation and Assembly of Quadruplex Nucleic Acids (2001) Hardin, C. C., Perry, A. G. and White, K. Biopolymers: Nucleic Acid Sciences 56, 147-194.
Textbooks:Cloning, Gene Expression and Protein Purification: Experimental Procedures and Process Rationale, Hardin, C. C., Edwards, J., Riell, A., Presutti, D., Miller, W. and Robertson, D. (2001) Oxford, University Press, N.Y. Experimental Biochemistry: Theory, Experiment, Analysis and Reporting (2005) Hardin, C. and Knopp, J., Sir Speedy Press; Oxford University Press, in review. Biochemical Bottom Lines, Hardin, C. C. (2005) Prentice-Hall, In review.
Awards:2003 Alumni Outstanding Teacher Award; Election to the NC Academy of Outstanding Teachers.
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