DNA-BINDING PROTEINS AS SITE-SPECIFIC NUCLEASES

DNA-binding proteins can be converted into site-specific nucleases by linking them to the chemical nuclease 1,10-phenanthroline-copper. This can be readily accomplished by converting a minor groove-proximal amino acid to a cysteine residue using site-directed mutagenesis and then chemically modifying the sulphydryl group with 5-iodoacetamido-1,10-phenanthroline-copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high-resolution nuclear magnetic resonance and X-ray crystal structures, and to locate binding sites of proteins within genomes.