title: Positively charged residues within the iron-sulfur cluster loop of E. coli MutY participate in damage recognition and removal.
abstract: Escherichia coli MutY is an adenine glycosylase involved in base excision repair 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 the DNA binding surface of MutY (Y. Guan et al., 1998, Nat. Struct. Biol. 5, 1058-1064). In order to investigate the role of specific residues within the FCL motif, we have determined the effects of replacing arginine 194, lysine 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 the properties of mutated enzymes in which lysine residues near the active site pocket were replaced with alanine or glycine. Substrate recognition was evaluated using a duplex containing a 2'-deoxyadenosine analog in a base pair opposite G or OG. These results indicate that removal of positively charged 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 enzyme exhibited a significant reduction (15-fold) of the rate of adenine removal from a G:A base pair-containing duplex. This is the first direct evidence 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 motif is intimately involved in the base removal process.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Chepanoske
authors.forename: M P
authors.initials: MP
authors.lastname: Golinelli
authors.forename: S D
authors.initials: SD
authors.lastname: Williams
authors.forename: S S
authors.initials: SS
authors.lastname: David
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001665:Binding Sites; D045647:DNA Glycosylases; D003839:Deoxyadenosines / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000737:chemistry; D013997:Time Factors
chemical_list: D003839:Deoxyadenosines; D013455:Sulfur; D007501:Iron; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D008239:Lysine; D000409:Alanine; C058118:2'-deoxyadenosine