SUNY at Albany
June 19-23, 2001
Biochemical evidence for base flip-out in DNA containing biologically-relevant levels of positive supercoiling
Cellular DNA is subjected to a variety of mechanical forces that affect DNA structure and dynamics. One such force is the torque applied to DNA during its passage through transcription and replication machinery. The loop organization of the human genome places topological constraints on the DNA such that strand separation caused by procession of an RNA or DNA polymerase generates twisting forces that unwind the helix behind the polymerase complex and over-wind the helix ahead of it (1). The resulting negative and positive supercoiling, respectively, has been detected as high levels of localized torsion in the 5'- and 3'-ends of genes (2,3).
We now present experimental results consistent with a model in which biologically relevant levels of positive supercoiling cause DNA bases to flip out of the helix. We have developed a technique that exploits archaeal histone proteins to generate positively supercoiled DNA molecules with a superhelical density of s = +0.05 (4). The over-wound DNA was found to be hyper-reactive toward dimethyl sulfate-induced alkylation of the N7-guanine (a site normally buried in the DNA helix) and N3-adenine, and to strand breaks produced by Cu(II)-mediated Fenton chemistry. These results suggest that over-winding of the helix causes increased solvent exposure of the bases. Compared to relaxed plasmid DNA, positively supercoiled DNA was also more reactive with agents selective for unpaired and exposed nucleobases, such as chloroacetaldehyde. Positively supercoiled DNA was also found to be more susceptible to cleavage by S1 nuclease, a single-strand endonuclease, than relaxed plasmid DNA.
On the basis of these results, we propose that biological levels of positive supercoiling lower the energy barrier to extrahelical excursion of DNA bases. This would lead to increased solvent exposure of the bases and explain the hyper-reactivity with base-directed chemicals, as well as the S1 nuclease sensitivity. Our results are consistent with the observations of Croquette and coworkers(5) who found that, at superhelical densities approaching s = 1, DNA undergoes a structural transition in which the sugar-phosphate backbone occupies the center of the helix and the bases are positioned extra-helically. The question of positive supercoiling-induced base flip-out as a stochastic process is under investigation. We are also investigating the effect of positive supercoiling on the kinetics and thermodynamics of base-flipping enzymes such as DNA glycosylases and methyl transferases.
References and Footnotes
Marita Barth and Peter Dedon,
Division of Bioengineering and Environmental Health,