Use of the hydroxyl radical and gel electrophoresis to study DNA structure Export

@article{3793, abstract = {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 5prime to 3prime direction along each adenine tract, with a minimum at the 3prime end of each adenine tract. The cleavage of the matching thy mine tract is similar, but the minimum in the pattern is offset in the 3prime 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. We discuss two problems in these areas which our lab has encountered and solutions we have found for them. Band compressions often occur in G/C-rich sequences. This can be avoided by adding 25-40 \% formamide to 50 \% urea denaturing polyacrylamide gels. Drying gels affords clearer autoradiographs of gels, yet higher percentage polyacrylamide gels are difficult to dry or even to remove from the plates. A method has been developed for drying 20-25 \% polyacrylamide gels that involves using plastic wrap, instead of filter paper, to remove the gel from the second plate.}, author = {Shafer, Gwen E. and Price, Mary A. and Tullius, Thomas D.}, citeulike-article-id = {3156107}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/elps.1150100518}, doi = {10.1002/elps.1150100518}, journal = {Electrophoresis}, keywords = {2r}, number = {5-6}, pages = {397--404}, posted-at = {2008-08-26 14:07:43}, priority = {2}, title = {Use of the hydroxyl radical and gel electrophoresis to study DNA structure}, url = {http://dx.doi.org/10.1002/elps.1150100518}, volume = {10}, year = {1989} }

TY - JOUR ID - 3793 L3 - citeulike-article-id:3156107 N2 - 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 5prime to 3prime direction along each adenine tract, with a minimum at the 3prime end of each adenine tract. The cleavage of the matching thy mine tract is similar, but the minimum in the pattern is offset in the 3prime 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. We discuss two problems in these areas which our lab has encountered and solutions we have found for them. Band compressions often occur in G/C-rich sequences. This can be avoided by adding 25-40 % formamide to 50 % urea denaturing polyacrylamide gels. Drying gels affords clearer autoradiographs of gels, yet higher percentage polyacrylamide gels are difficult to dry or even to remove from the plates. A method has been developed for drying 20-25 % polyacrylamide gels that involves using plastic wrap, instead of filter paper, to remove the gel from the second plate. IS - 5-6 JF - Electrophoresis EP - 404 TI - Use of the hydroxyl radical and gel electrophoresis to study DNA structure VL - 10 SP - 397 KW - 2r AU - Shafer, Gwen AU - Price, Mary AU - Tullius, Thomas PY - 1989/// UR - http://dx.doi.org/10.1002/elps.1150100518 ER -