Psoralen cross-linking as probe of torsional tension and topological domain size in vivo

DNA within a cell is organized with unrestrained torsional tension, and eac h molecule is divided Into multiple individual topological domains. Psorale n photobinding can be used as an assay for supercoiling and topological dom ain size in living cells. Psoralen photobinds to DNA at a rate nearly linea rly proportional to superhelical density. Comparison of the rate of photobi nding to supercoiled and relaxed DNA in cells provides a measure of superhe lical density. For this, in vivo superhelical tension is relaxed by the int roduction of nicks by either ionizing radiation or photolysis of bromodeoxy uridine in the DNA. Since nicks are introduced in a random fashion, the dis tribution of nicks is described by a Poisson distribution. Thus, after nick ing, the fraction of topological domains containing no nicks is described b y the zero term of the Poisson distribution. From measurement of the number of nicks introduced in the DNA and the fraction of torsional tension remai ning, an average topological domain size can be estimated. Using this logic , procedures were designed and described for measuring supercoiling and dom ain size at specific sites in eukaryotic genomes. (C) 1999 Academic Press.