A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: Application to the EcoRI and Rsrl restriction and modification enzymes

Abstract

A modified ollgodeoxyribonucleotide duplex containing an unnatural internucleotlde trisubstltuted 3′ to 5′ pyrophosphate bond in one strand [5′(oligo1)3′-P(OCH3) P-5′(oligo1) 3′] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [-P-5′(oligo2)3′] becomes attached to an amino acid nucleophlllc group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophllic groups at the active sites of the EcoRI and Rsrl restriction and modification enzymes with an ollgodeoxyribonucleotide duplex containing a modified sclssile bond in the EcoRI recognition site. With the EcoRI and Rsrl endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the Rsrl methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3′-phosphate of the modified pyrophosphate in the substrate and a nucleophllic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3′-0-methylphosphate [5′(oligo1)3′-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein- DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase. © 1992 Oxford University Press.