Positively charged residues within the iron-sulfur cluster loop of E-coli MutY participate in damage recognition and removal

Escherichia coli MutY is an adenine glycosylase involved in base excision r epair that recognizes OG:A (where OG = 7,8-dihydro-8-oxo-2'-deoxyguanosine) and G:A mismatches in DNA MutY contains a solvent-exposed polypeptide loop between two of the cysteine ligands to the [4Fe-4S](2+) cluster, referred to as the iron-sulfur cluster loop (FCL) motif. The FCL is located adjacent to the proposed active site pocket and has been suggested to be part of th e DNA binding surface of MutY (Y. Guan et al, 1998, Naf. Struct. Biol, 5, 1 058-1064). In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysin e 196, and lysine 198 with alanine on the enzymatic properties of MutY. The properties of the R194A, K196A, and K198A enzymes were also compared to th e properties of mutated enzymes in which lysine residues near the active si te pocket mere replaced with alanine or glycine. Substrate recognition was evaluated using a duplex containing a S'-deoxyadenosine analog in a base pa ir opposite G; or OG;, These results indicate that removal of positively ch arged amino acids within the FCL and the active site compromise the ability of the enzyme to bind to the substrate analog. However, only the K198A enz yme exhibited a significant reduction (15-fold) of the rate of adenine remo val from a G:A base pair-containing duplex. This is the first direct eviden ce that Lys 198 within the FCL motif of MutY has a role in specific damage recognition and removal. Furthermore, these results suggest that the FCL mo tif is intimately involved in the base removal process. (C) 2000 Academic P ress.