Book of Abstracts: Albany 2005
The Effect of a Single-Nucleoside Gap in DNA on Cyclization Kinetics
It is known that oxidizing agents and ionizing radiation damage DNA by producing gaps in a DNA duplex; however, a better understanding of the details of how the cell?s repair system recognizes these gaps is needed. Thus, studying the structural and biophysical properties of gapped DNA may lead to new information on the recognition of DNA strand gaps by repair proteins. Previous experiments from our laboratory have shown that a gap in one strand of a DNA duplex represents a site of anisotropic bending (1). This finding leads one to ask whether such a lesion might also be torsionally flexible. To gain further insight into the dynamics of gapped DNA, cyclization assays were conducted on restriction fragments of various lengths. Cyclization of linear DNA can be used to measure its torsional and bending flexibility (2). In our experiments we expose a DNA restriction fragment to hydroxyl radical treatment to generate a library of gapped DNA molecules. The sticky ends on the restriction fragment are then treated with T4 DNA ligase to form either circles or linear multimers. The single-nucleoside gap can create a site of localized flexibility and affect the cyclization probability of the DNA strand. If cyclization were to occur without dependence on the phase of the strand gap relative to a fixed bend we have introduced into the DNA molecule, it would imply that a strand gap is a site of torsional flexibility. Conversely, cyclization with phase dependence would imply that a gap is torsionally stiff. Ultimately, our experiments are designed to distinguish between three possible outcomes for the structure and dynamics of gapped DNA: (i) bent, torsionally flexible; (ii) bent, torsionally stiff; or (iii) torsionally flexible, unbent. Thus, important aspects of the structure and dynamics of a DNA strand gap can be determined from the results of these experiments.
References and Footnotes
Truc T. Diep*