TRANSFORMING THE ESCHERICHIA-COLI TRP-REPRESSOR INTO A SITE-SPECIFIC NUCLEASE

The Escherichia coli Trp repressor has been converted into an operator -specific nuclease by alkylating cysteine-49, inserted by site-directe d mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline. In the pres ence of copper ion and thiol, high yields (>50%) of double-stranded br eaks of DNA are observed after a 20-min reaction. The high cleavage ef ficiency of this derivatized protein (Trp repressor E49C-OP) can be at tributed to the proximity of cysteine-49 to the minor groove, the site of the C-1H of the deoxyribose and the target of the oxidative nuclea se activity of (1,10-phenanthroline)copper. Since sequence position 49 is close to the protein's C2 dyad axis and adjacent to the minor groo ve, Trp repressor E49C-OP reacts with the operator DNA near the bindin g site of this symmetry locus of the protein. The patterns of scission of the trpR, aroH, and trpEDCBA operators (a) confirm the orientation of the repressor to the operator predicted from the X-ray study of a cocrystal (Otwinowski et al., 1988) and (b) support the model for tand em binding of the repressor to the trpR, aroH, and trpEDCBA operators based on DNase I footprinting and methylation interference (Kumamoto e t al., 1987). There are one, two, and three binding sites for the repr essor on the trpR, aroH, and trpEDCBA operators, respectively. In addi tion to providing a novel approach to studying the interactions of DNA binding proteins, 1,10-phenanthroline-derivatized proteins such as Tr p repressor E49C-OP may be useful as rare cutters in the analysis of h igh molecular weight DNAs, especially if their binding specificities c an be altered.