A single engineered point mutation in the adenine glycosylase MutY confersbifunctional glycosylase/AP lyase activity

The E. coli adenine glycosylase MutY is a member of the base excision repai r (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunc tional glycosylase/AP lyases bear a conserved lysine group in the active si te region, which is believed to be the species performing the initial nucle ophilic attack at C1' in the catalysis of base removal. Monofunctional glyc osylases such as MutY are thought to perform this C1' nucleophilic displace ment by a base-activated water molecule, and, indeed, the conservation of a mine functionality positioning has not been observed in protein sequence al ignments. Bifunctional glycosylase/AP lyase activity was successfully engin eered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine e xcision for both G:A and OG:A mispair substrates. The extent of DNA backbon e cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Im portantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in th e glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechan isms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to conv ert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.