Gapped DNA is anisotropically bent.
Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.
Guo
Hong
H
Protometrix, Inc., 66 High Street, Guilford, CT 06437, USA.
Tullius
Thomas D
TD
D016880:Anisotropy; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006878:Hydroxides; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010957:Plasmids / Q000737:chemistry*; D011089:Polydeoxyribonucleotides / Q000737:chemistry*
D006878:Hydroxides; D011089:Polydeoxyribonucleotides; D004247:DNA; C031356:hydroxide ion