Chapter 14 Protein—DNA Cross-Linking as a Means to Determine the Distribution of Proteins on DNA in Vivo

The protein-DNA cross-linking can be used to determine the distribution of proteins on DNA in vivo. The temporal and spatial distributions of particular proteins on specific DNA sequences provide key insights into the regulation and mechanics of transcription and chromosome replication and basic information on chromatin structure. The chapter describes a method for directly mapping the distribution of a protein on DNA in living cells by ultraviolet (UV)-induced cross-linking. UV irradiation of cells cross-links protein to DNA at the points of contact. The protein–DNA adducts are purified from cells, and then the protein of interest is immunoprecipitated with an appropriate antibody. The distribution of the protein on DNA in the cell is revealed by identifying specific restriction fragments that coprecipitate with the protein via their UV-induced covalent attachment to the protein. The UV-cross-linking method provides a unique opportunity to evaluate mechanistic questions in vivo. The method has been used to demonstrate that Drosophila topoisomerase I is recruited during heat shock to activated transcription units. It has also provided a direct measure of the relative in vivo density of RNA polymerase II on a variety of Drosophila genes.