Use of the hydroxyl radical and gel electrophoresis to study DNA structure.
The hydroxyl radical has been used as a chemical probe to study in solution the structure of DNA and DNA-protein complexes. The hydroxyl radical abstracts a deoxyribose hydrogen atom, cleaving one strand of the DNA. The cutting pattern, visualized by separating the cleavage products using gel electrophoresis, shows the reactivity of each backbone position toward the radical. This method has been applied to studies of DNA bending and helical twist. Phased runs of adenines (adenine tracts) cause sequence-directed DNA bending. The hydroxyl radical cleavage of a bent DNA fragment containing short adenine tracts phased with the helix screw gives rise to an unusual cutting pattern. The hydroxyl radical cleavage rate decreases in the 5' to 3' direction along each adenine tract, with a minimum at the 3' end of each adenine tract. The cleavage of the matching thymine tract is similar, but the minimum in the pattern is offset in the 3' direction. This pattern on the autoradiograph of the gel is interpreted to indicate that bending is accompanied by a narrow minor groove in the DNA molecule. Furthermore, hydroxyl radical cleavage results in different cutting patterns for two similar sequences, (CGA4T4)5 and (CGT4A4)5, which have been shown to be bent and relatively straight, respectively. The hydroxyl radical method has also been used to determine the helical repeat of the metallothionein IIA gene to be about 10.5 base pairs per turn. Methods of optimizing the hydroxyl radical reaction for DNA-protein footprinting are discussed. Because individual gel bands give information about cutting frequency at particular positions in the backbone, gel resolution and clear autoradiographs are important to this work.(ABSTRACT TRUNCATED AT 250 WORDS)
Shafer
G E
GE
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Price
M A
MA
Tullius
T D
TD
D001345:Autoradiography; D002130:Calcium Phosphates; D004269:DNA, Bacterial / Q000032:analysis*; D003720:Densitometry; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005296:Ferrous Compounds; D006878:Hydroxides; D008668:Metallothionein / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009710:Nucleotide Mapping
D002130:Calcium Phosphates; D004269:DNA, Bacterial; D005296:Ferrous Compounds; D006878:Hydroxides; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C055526:Fe(II)-EDTA; C485838:calcium phosphate, monobasic, anhydrous; D008668:Metallothionein; C020243:calcium phosphate; D004492:Edetic Acid; C485829:calcium phosphate, dibasic, anhydrous