Nucleic Acids Research, 1992, Vol. 20, No. 14 3713-3719
© 1992
ENZYMOLOGY |
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
Department of Chemistry, Moscow State University Moscow 119899, Russia 1Department of Biochemistry, College of Medicine, University of Illinois Urbana, IL 61801, USA
*To whom correspondence should be addressed
Received March 16, 1992. Revised June 10, 1992. Accepted June 10, 1992.
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.
+Present address: University of Vermont, Department of Microbiology, Given Building, Burlington, VT 05405, USA
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