The use of micrococcal nuclease as a probe for drug-binding sites on DNA

The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.