The chapter describes the protein–DNA interactions in vivo by examining genes in Saccharomyces cerevisiae and Drosophila melanogaster by chromatin footprinting. The interactions of proteins with chromosomal DNA control a variety of cellular processes, including gene transcription, DNA packing, replication, recombination, and DNA repair. Specific cloned DNA sequences can be assembled with proteins isolated from cellular extracts to provide details on the binding of each factor and its effect on the overall structure and activity of the complex. One high-resolution method of mapping DNA–protein contacts that can be used with both reconstituted and cellular assemblies is DNA footprinting. This technique is designed to locate the binding sites of purified proteins on end-labeled DNA fragments by determining which DNA base pairs become selectively resistant to DNA cleavage reagents. The in vitro footprinting provides a high-resolution contact map with protein components that can be defined biochemically and assembled stepwise. The chromatin footprinting is most effective when used in parallel with biochemical studies as a predictive or confirmatory tool. The chapter discusses several viable alternatives and their strengths and weaknesses along with the protocols used to map DNase I sensitivity in Saccharomyces cerevisiae and methidium propyl-EDTA.Fe(II) sensitivity in Drosophila melanogaster.
All Science Journal Classification (ASJC) codes
- Cell Biology