FUNCTION OF THE ZINC-FINGER IN ESCHERICHIA-COLI FPG PROTEIN

Fpg protein of Escherichia coli cleaves duplex DNA containing the oxid atively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., S hibutani, S., Chung, M.-H., Laval, J., Grollman, A. P., and Nishimura, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4690-4694). This DNA re pair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Co nnor, T. R., Lederer, F., Gougette, A., and Laval, J. (1990) J. Biol. Chem. 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lya se activities are physically associated. Amino acid sequence analysis reveals a putative single zinc finger motif of the CC/CC type located near the carboxyl terminus. A gel mobility shift assay was used to ass ay binding of Fpg protein to a non-cleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran res idue. High resolution hydroxyl radical DNA footprinting showed protect ion centered around the tetrahydrofuran residue. No footprint was obse rved on the complementary strand. To establish the role of COOH-termin al zinc finger in DNA binding and/or DNA cleavage, amino acid substitu tions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber). In addition, a double amino acid substitution w as generated at Cys-244 and Cys-247 (C244S/C247S). These mutant Fpg pr oteins lack DNA binding or cleavage activity, as tested in crude lysat es of Escherichia coli. Wild type Fpg protein contains one zinc/protei n molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, a s measured by atomic absorption spectroscopy. This mutation did not si gnificantly alter secondary structure, as assessed by circular dichroi sm spectroscopy. Our results suggest that Fpg protein utilizes its sin gle COOH-terminal zinc finger motif in DNA binding.