The long-term goals of this project are to synthesize and study nonpolar, non-hydrogen-bonding nucleosides and nucleotides as biophysical probes of noncovalent bonding in DNA-DNA, protein-nucleotide, and protein-DNA interactions. These molecules are designed to mimiC as closely as possible the shape and size of the natural nucleosides, but without polar hydrogen bonding N, N-H and C=O groups. Preliminary studies of several such analogs over the past 1.5 years have shown that they display a number of unusual properties both alone and in the context of DNA helices, and they have already led to a number of surprising and valuable findings in the study of stacking and pairing in duplex DNA and in the study of the fidelity of Klenow fragment of E. Coli DNA Polymerase I. The stacking results are likely to be of practical use in the design of helix-stabilizing agents for oligonucleotide-based therapeutics, and the polymerase studies will aid in the design of novel polymerase inhibitors (with potential antiviral or antineoplastic effects). In the long term, it is hoped that these new analogs will be of general utility in biophysical studies of noncovalent bonding. The proposed studies are aimed at the use of these novel analogs in three separate biomolecular systems, both as a test of the properties and utility of the compounds, and also to shed new light on noncovalent interactions in these systems. Experiments are planned for the study of base stacking in DNA, noncovalent bonding requirements in nucleotide incorporation by DNA polymerases, and energetic contributions to DNA binding by TATA-binding proteins. Specific plans for the term covered by this proposal include: (1) ln-depth study of the six current analogs (and their natural counterparts) as probes of thermodynamic factors of base stacking in DNA; (2) Synthesis of several new analogs to gain additional data in stacking studies and in polymerase experiments; (3) In-depth study of the processing of these analogs by E. Coli Pol I and three other polymerases; (4) Use of these analogs to test energetics of specific interactions between yeast TATA-binding protein and a consensus TATA recognition sequence.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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University of Rochester
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Maki, Angele S; Kim, TaeWoo; Kool, Eric T (2004) Direct comparison of A- and T-strand minor groove interactions in DNA curvature at A tracts. Biochemistry 43:1102-10
Lai, Jacob S; Kool, Eric T (2004) Selective pairing of polyfluorinated DNA bases. J Am Chem Soc 126:3040-1
Gao, Jianmin; Liu, Haibo; Kool, Eric T (2004) Expanded-size bases in naturally sized DNA: evaluation of steric effects in Watson-Crick pairing. J Am Chem Soc 126:11826-31
Francis, Anthony W; Helquist, Sandra A; Kool, Eric T et al. (2003) Probing the requirements for recognition and catalysis in Fpg and MutY with nonpolar adenine isosteres. J Am Chem Soc 125:16235-42
Lai, Jacob S; Qu, Jin; Kool, Eric T (2003) Fluorinated DNA bases as probes of electrostatic effects in DNA base stacking. Angew Chem Int Ed Engl 42:5973-7
Liu, Haibo; Gao, Jianmin; Lynch, Stephen R et al. (2003) A four-base paired genetic helix with expanded size. Science 302:868-71
Delaney, James C; Henderson, Paul T; Helquist, Sandra A et al. (2003) High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds. Proc Natl Acad Sci U S A 100:4469-73
Rausch, Jason W; Qu, Jin; Yi-Brunozzi, Hye Young et al. (2003) Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection. Proc Natl Acad Sci U S A 100:11279-84
Maki, Angele; Brownewell, Floyd E; Liu, Dongyu et al. (2003) DNA curvature at A tracts containing a non-polar thymine mimic. Nucleic Acids Res 31:1059-66
Washington, M Todd; Helquist, Sandra A; Kool, Eric T et al. (2003) Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta. Mol Cell Biol 23:5107-12

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