SITE-DIRECTED MUTAGENESIS OF GLUTAMIC-ACID-172 TO GLUTAMINE COMPLETELY INACTIVATED HUMAN O-6-ALKYLGUANINE-DNA-ALKYLTRANSFERASE

DNA repair by O-6-alkylguanine-DNA-alkyltransferase involves the stoic hiometric transfer of the O-6-alkyl group from the guanine lesion to t he active-site cysteine residues of the protein. Site-directed mutagen esis of glutamic acid 172 of human O-6-alkylguanine-DNA-alkyltransfera se (EC 2.1.1.63) to glutamine totally abolished the alkyltransferase a ctivity of the protein. This suggests that glutamic acid 172 is crucia l to the alkyl transfer. It may act as a general acid (as CO2H) or bas e (as CO2-), or have a role as a component of a salt-link (-CO2-.....N -+-), vital for the structural integrity of the active site. This is t he first mutational inactivation of a protein in this family of DNA re pair molecules by means of a residue change outside the highly conserv ed pentet (PCHRV) which includes the active-site cysteine. (C) 1994 Ac ademic Press, Inc.