FPG PROTEIN OF ESCHERICHIA-COLI IS A ZINC FINGER PROTEIN WHOSE CYSTEINE RESIDUES HAVE A STRUCTURAL AND OR FUNCTIONAL-ROLE

The Fpg protein of Escherichia coli is a DNA repair enzyme with DNA gl ycosylase, abasic site nicking, and deoxyribose excising activities. A nalysis of the amino acid sequence of this protein suggests that the F pg protein is a zinc finger protein with a Cys-X2-Cys-X16-Cys-X2-Cys m otif. Competition experiments show that the Fpg protein substitutes Cu (II), Cd(II), and Hg(II), metal ions classically associated with subst itutions in zinc finger proteins. The Fpg protein activities are inhib ited following the reaction with a Cys-specific reagent at low protein :reagent ratios, suggesting that these residues are important for the enzymatic activities. Site-directed mutagenesis was used to produce 6 mutant Fpg proteins with Cys --> Gly mutations. Substitution of the zi nc in these proteins by Zn-65(II) indicates that all the proteins bind zinc, but the Zn(II) is not retained as strongly in the zinc finger m utants. The mutations in the Fpg protein outside the zinc finger conse nsus sequence do not eliminate the Fapy-DNA glycosylase and abasic sit e nicking. One of the Fpg mutant proteins outside the zinc finger has a reduced capacity to release deoxyribose from abasic sites. Cys --> G ly mutations in the zinc finger consensus sequence reduce all three af orementioned activities substantially. The purified Fpg proteins with Cys --> Gly mutations in the zinc finger consensus sequence do not inc ise DNA at abasic sites with the same efficiency nor mechanism as the native Fpg protein. The wild type Fpg protein and the Fpg proteins mut ated outside the zinc finger sequence bind an oligonucleotide with a u nique chemically reduced abasic site in a defined sequence as assayed by retention on nitrocellulose filters, whereas the mutant Fpg protein s within the zinc finger sequence do not bind to the same oligonucleot ide. Therefore, the disruption of zinc coordination in the zinc finger of the Fpg protein is associated with decreased binding capacity to D NA as well as decreased enzymatic activities.