Dynamic Profiles of DNA: Analysis of CAP- and LexA Protein-Binding Regions with Endonucleases

Abstract

We examined ssDNA and dsDNA containing a CAP-binding region or a lexA protein-binding region in therecA gene of Escherichia coli with endonucleases (E. coli endonuclease I and bovine DNase I) to deduce the structural conformation of dsDNA as well as ssDNA. Each nuclease produced its own cleavage pattern. Some cleavages occurred at common sites in ssDNA and dsDNA. The common cleavage sites for endonuclease I included ones that have been inferred to be functionally important. Efficient cleavage occurred at bent sites in the CAP-binding region, and at "SOS box" sites in the recA gene. NTP (dNTP) greatly increased the cleavage at these sites in the ssDNA and dsDNA. Next, we investigated whether mutations which affect function can be ascribed to variation in the conformation of DNA. Polynucleotides containing a single base substitution derived from the mutants in the CAP-binding region were cleaved by endonuclease I. The cleavage pattern varied predominantly around the substituted nucleotide in dsDNA. Thus, we confirmed that DNA structure is closely related to function. Complexes of endonuclease I and synthetic polynucleotides that had one cleavage site were confirmed to exist in the absence of magnesium ions by gel-shift assay.