Psoralen Cross-Linking as Probe of Torsional Tension and Topological Domain Sizein Vivo

Abstract

DNA within a cell is organized with unrestrained torsional tension, and each molecule is divided into multiple individual topological domains. Psoralen photobinding can be used as an assay for supercoiling and topological domain size in living cells. Psoralen photobinds to DNA at a rate nearly linearly proportional to superhelical density. Comparison of the rate of photobinding to supercoiled and relaxed DNA in cells provides a measure of superhelical density. For this,in vivosuperhelical tension is relaxed by the introduction of nicks by either ionizing radiation or photolysis of bromodeoxyuridine in the DNA. Since nicks are introduced in a random fashion, the distribution of nicks is described by a Poisson distribution. Thus, after nicking, the fraction of topological domains containing no nicks is described by the zero term of the Poisson distribution. From measurement of the number of nicks introduced in the DNA and the fraction of torsional tension remaining, an average topological domain size can be estimated. Using this logic, procedures were designed and described for measuring supercoiling and domain size at specific sites in eukaryotic genomes.