CLEAVAGE AND BINDING OF A DNA FRAGMENT CONTAINING A SINGLE 8-OXOGUANINE BY WILD-TYPE AND MUTANT FPG PROTEINS

A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding prope rties of the Fpg protein from E.coli. The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for dup lexes where 8-OxoG was opposite C (G/C) or T (*G/T). In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (G/G) and A (G/A) were 5-fold and 200-fold slower. Gel retardation studies sho wed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where t he 8-OxoG was opposite purines. K(D)app values were 0.6 nM (G/C), 1.0 nM (G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A). The Fpg protein also bin ds to unmodified (G/C) duplex and a K(D)app of 90 nM was measured. The cleavage and binding of the (G/C) duplex were also studied using bac terial crude lysates. Wild type E.coli crude extract incised the 8-Oxo G containing strand and formed a specific retardation complex with the (G/C) duplex. These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial str ain whose fpg gene was inactivated. Furthermore, we have studied the p roperties of 6 mutant Fpg proteins with Cys --> Gly mutations. The res ults showed that the 2 Fpg proteins with Cys --> Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant. In contrast, the 4 Fpg proteins with Cys --> Gly mutations within the zinc finger motif neither cleave nor bind the ( G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotyp e.