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An efficient NMR experiment for analyzing sugar-puckering in unlabeled DNA: application to the 26-kDa dead ringer-DNA
J Iwahara1, J M Wojciak, R T Clubb
1Department of Chemistry and Biochemistry, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California, Los Angeles, 405 Hilgard Avenue, California 90095-1570, USA. iwahara@mbi.ucla.edu
Journal of Magnetic Resonance (San Diego, Calif. : 1997)
|December 13, 2001
Summary
We developed a new NMR experiment to determine DNA sugar puckering in large molecules. This method reveals DNA adopts an S-type configuration when bound to the Dead Ringer protein.
Area of Science:
- Biochemistry
- Structural Biology
- Nuclear Magnetic Resonance (NMR) Spectroscopy
Background:
- Estimating sugar puckering in high-molecular-weight DNA is crucial for understanding DNA structure and protein-DNA interactions.
- Traditional NMR methods face challenges with unlabeled, large DNA molecules.
- Characterizing DNA conformation in protein-DNA complexes requires specialized techniques.
Purpose of the Study:
- To introduce a novel NMR experiment for analyzing sugar puckering in unlabeled, high-molecular-weight DNA.
- To enable the study of DNA conformation within protein-DNA complexes.
- To provide an accessible method for estimating deoxyribose ring puckering.
Main Methods:
- A new NMR experiment combining NOESY (Nuclear Overhauser Effect Spectroscopy) with a constant-time scalar coupling period.
- Two subexperiments varying the duration of (3)J(H3'H2') and (3)J(H3'H2") couplings on H3' magnetization.
- Implementation of isotope filters to remove signals from (13)C-labeled proteins, facilitating analysis of protein-DNA complexes.
Main Results:
- The experiment allows for easy analysis by comparing NOE cross-peak signal intensities from H3' atoms.
- The sum of (3)J(H3'H2') and (3)J(H3'H2") couplings can be estimated, directly correlating to deoxyribose ring puckering.
- Demonstrated utility on a 26-kDa Dead Ringer protein-DNA complex, showing uniformly S-type DNA configuration upon binding.
Conclusions:
- The new NMR experiment effectively estimates sugar puckering in high-molecular-weight unlabeled DNA.
- This method is particularly useful for analyzing unlabeled DNA within labeled protein-DNA complexes.
- DNA bound to the Dead Ringer protein adopts a consistent S-type sugar puckering configuration.
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