Function of the zinc finger in Escherichia coli Fpg protein.
Fpg protein of Escherichia coli cleaves duplex DNA containing the oxidatively damaged base 8-oxo-7,8-dihydroguanine (Tchou, J., Kasai, H., Shibutani, 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 repair enzyme contains one zinc atom/protein molecule (Boiteux, S., O'Connor, T. R., Lederer, F., Gougette, A., and Laval, J. (1990) J. Biol. Chem. 265, 3916-3922); its N-glycosylase and apurinic/apyrimidinic lyase 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 assay binding of Fpg protein to a noncleavable substrate analog, namely an oligodeoxynucleotide duplex containing a single tetrahydrofuran residue. High resolution hydroxyl radical DNA footprinting showed protection centered around the tetrahydrofuran residue. No footprint was observed on the complementary strand. To establish the role of COOH-terminal zinc finger in DNA binding and/or DNA cleavage, amino acid substitutions and an amber mutation were introduced at Cys-244 (C244S, C244H, C244A, and C244amber). In addition, a double amino acid substitution was generated at Cys-244 and Cys-247 (C244S/C247S). These mutant Fpg proteins lack DNA binding or cleavage activity, as tested in crude lysates of Escherichia coli. Wild type Fpg protein contains one zinc/protein molecule, whereas the mutant Fpg protein (C244S/C247S) lacks zinc, as measured by atomic absorption spectroscopy. This mutation did not significantly alter secondary structure, as assessed by circular dichroism spectroscopy. Our results suggest that Fpg protein utilizes its single COOH-terminal zinc finger motif in DNA binding.
Tchou
J
J
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Michaels
M L
ML
Miller
J H
JH
Grollman
A P
AP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D016335:Zinc Fingers / Q000502:physiology*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D017665:Hydroxyl Radical; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli