The objective of this proposal is the further development and application of a spin assay for nucleic acid binding proteins. The assay provides a rapid, sensitive, and safe (no radiolabeling) means for detecting complexes between nucleic acids and nucleic acid binding proteins by electron spin resonance (ESR) without potential interference by a matrix such as filters or gels. The complexes are detected with lst and 2nd generation spin labeled nucleic acids through nitroxide radicals as reporter groups which are incorporated at random to a small extent (nitroxide/nucleotide varies between 0.002 to 0.02 into the nucleic acids). 1st generation spin labeled nucleic acids are labeled non-site specifically by chemical means, whereas 2nd generation spin labeled nucleic acids (labeling specificity with respect to nucleotide) are prepared enzymatically. It is proposed to also include 3rd generation spin labeled nucleic acids (labeling specificity with respect to nucleotide and sequence) in the spin assay. The spin assay employing these spin labeled nucleic acids will be used either to detect binding of a particular protein to a given nucleic acid under a variety of conditions (protein modification, salt concentration, etc.) or to determine the relative affinity of a variety of nucleic acids for a particular protein through competition experiments with spin labeled and unlabeled nucleic acids. Recently developed computer programs will be used to determine the fraction F of nucleic acids in the complexed state directly from the ESR spectrum. The assay will be applied to follow the binding of gene 32 and gene 5 proteins to nucleic acids, the binding of nucleic acid inhibitors and their analogs to AMV reverse transcriptase, and to further define the role of gene 32 protein and SSB protein of E. coli during the replicative process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027002-10
Application #
3274451
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1979-07-01
Project End
1990-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Keyes, R S; Bobst, E V; Cao, Y Y et al. (1997) Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels. Biophys J 72:282-90
Bobst, E V; Keyes, R S; Cao, Y Y et al. (1996) Spectroscopic probe for the detection of local DNA bending at an AAA triplet. Biochemistry 35:9309-13
Keyes, R S; Cao, Y Y; Bobst, E V et al. (1996) Spin-labeled nucleotide mobility in the boundary of the EcoRI endonuclease binding site. J Biomol Struct Dyn 14:163-72
Strobel, O K; Keyes, R S; Sinden, R R et al. (1995) Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance. Arch Biochem Biophys 324:357-66
Keyes, R S; Bobst, A M (1995) Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry 34:9265-76
Keyes, R S; Bobst, A M (1993) A comparative study of Scatchard-type and linear lattice models for the analysis of EPR competition experiments with spin-labeled nucleic acids and single-strand binding proteins. Biophys Chem 45:281-303
Bobst, E V; Perrino, F W; Meyer, R R et al. (1991) An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli. Biochim Biophys Acta 1078:199-207
Strobel, O K; Kryak, D D; Bobst, E V et al. (1991) Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization. Bioconjug Chem 2:89-95
Strobel, O K; Keyes, R S; Bobst, A M (1990) Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry 29:8522-8
Strobel, O K; Keyes, R S; Bobst, A M (1990) An electron paramagnetic resonance probe to detect local Z-DNA conformations. Biochem Biophys Res Commun 166:1435-40

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