Relating independent measures of DNA curvature: electrophoretic anomaly and cyclization efficiency.
Electrophoretic methods are often used to measure DNA curvature and protein-induced DNA bending. Though convenient and widely-applied, quantitative analyses are generally limited to assays for which empirical calibration standards have been developed. Alternatively, solution-based cyclization of short DNA duplexes allows analysis of DNA curvature and bending from first principles, but a detailed understanding of this assay is still lacking. In this work, we demonstrate that calibration with an independent electrophoretic assay of DNA curvature permits interpretation of cyclization assay results in a quantitatively meaningful way. We systematically measure intrinsic DNA curvature in short duplexes using a well-established empirical ligation ladder assay. We then compare the results to those obtained from the analysis of the distribution of circular products obtained in simple enzymatic cyclization assays of the same duplexes when polymerized. A strong correlation between DNA curvature estimates from these two assays is obtained for DNA fragments between 150-300 bp in length. We discuss how this result might be used to improve quantitative analysis of protein-mediated bending events evaluated by cyclization methods. Our results suggest that measurements of DNA curvature obtained under similar conditions, in solution and in an acrylamide gel matrix, can be compared directly. The ability to correlate results of these simple assays may prove convenient in monitoring DNA curvature and flexibility.
Hardwidge
P R
PR
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, MN 55905, USA.
Den
R B
RB
Ross
E D
ED
Maher
L J
LJ
D001483:Base Sequence; D002138:Calibration; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005092:Exonucleases / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry
D009841:Oligonucleotides; D004247:DNA; D005092:Exonucleases; D008274:Magnesium