Unique 31P spectral response to the formation of a specific restriction enzyme-DNA complex.
Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme-DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is Pvull endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here 31P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The 31P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of 31P resonances shift dramatically. The magnitudes of the chemical shifts (2-3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct 31P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation.
Dupureur
Cynthia M
CM
Department of Chemistry and Biochemistry, One University Blvd., University of Missouri-St. Louis, St. Louis, MO 63121, USA. cdup@umsl.edu
D000577:Amides / Q000737:chemistry; D003460:Crystallization; D004247:DNA / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D009841:Oligonucleotides / Q000737:chemistry; D010756:Phosphoric Acids / Q000737:chemistry; D010759:Phosphorus Isotopes; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D000577:Amides; D009841:Oligonucleotides; D010756:Phosphoric Acids; D010759:Phosphorus Isotopes; D004247:DNA; C011067:phosphoramidic acid; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific