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 properties 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 duplexes 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 showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines. KDapp 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 binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured. The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates. Wild type E. coli crude extract incised the 8-OxoG 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 strain whose fpg gene was inactivated. Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations. The results 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 phenotype.
Castaing
B
B
Laboratoire de Biologie Structurale du CNRS, Gif sur Yvette, France.
Geiger
A
A
Seliger
H
H
Nehls
P
P
Laval
J
J
Zelwer
C
C
Boiteux
S
S
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005998:Glycine / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016335:Zinc Fingers
D029968:Escherichia coli Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D003545:Cysteine; D005998:Glycine