Book of Abstracts: Albany 2003
June 17-21 2003
Measuring Protein-induced DNA Bending by Cyclization of Short DNA Fragments
The efficiency of cyclization of short DNA is extremely sensitive to intrinsic or induced bends of the double helix and can be used to measure the bend angle induced by protein binding. However, DNA rigidity normally prevents cyclization in the undisturbed complexes. Also, cyclization of a short fragment requires proper accounting for the torsional orientation of its ends, which necessitates series of laborious measurements. To overcome these problems we developed a modification of the method based on cyclization of DNA fragment containing a single-stranded gap near one of its ends. The gap serves as a hinge for lateral and torsional deflections of the double helix, since single-stranded DNA is very flexible. The measurement for a single length of such fragment is sufficient to estimate the value of the bend angle. We developed a simple procedure to obtain DNA molecules with a single-stranded gap and performed quantitative testing of the approach.