Substrate specificity of Fpg protein. Recognition and cleavage of oxidatively damaged DNA.

J Tchou,V Bodepudi,S Shibutani,I Antoshechkin,J Miller,A.P Grollman,F Johnson

doi:10.1016/s0021-9258(17)36608-5

Abstract

The 8-oxoguanine-DNA glycosylase of Escherichia coli, also known as formamidopyrimidine-DNA glycosylase (Fpg protein), has N-glycosylase and AP-lyase activities. This enzyme repairs oxidative DNA damage by efficiently removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA. Defined oligodeoxynucleotides containing various 8-oxopurines were used to examine the substrate specificity of Fpg protein and to establish the role of functional groups in DNA on damage recognition and catalysis. Binding affinities of Fpg protein were established for duplex oligodeoxynucleotides containing 8-oxo-2'-deoxyguanine, 8-oxo-2'-deoxyadenine, 8-oxo-2'-deoxynebularine, 8-oxo-2'-deoxyinosine, abasic sites, and a ring-open adduct of C8-aminofluorene guanine. The C8 keto group of 8-oxodG:dC presents in the major groove and is correlated with tight binding (Kd = 8.9 nM). Binding is much weaker when the C8 keto functional group is in the minor groove, as in 8-oxodG:dA (Kd = 340 nM). Km and Vmax were determined for the cleavage reaction. Specificity constants (Kcat/Km) are consistently higher for oligodeoxynucleotide duplexes containing 8-oxopurines with C6 and C8 keto groups, as in 8-oxodG:dC and 8-oxodI:dC, where Kcat/Km are 9.3 and 18 min-1 nM x 10(-3), respectively. 8-oxodN:dC lacks the C6 keto group; the specificity constant is 0.024 min-1 nM x 10(-3). Taken together, our data suggest that the C8 keto group of 8-oxodeoxyguanine and the carbonyl moiety of formamidopyrimidine enable Fpg protein to recognize and bind duplex DNA containing these modified bases. An enzyme-catalyzed reaction involving the C6 keto group of the substrate leads to removal of these lesions. A mechanism involving protonation at O-6 of 8-oxoguanine is proposed to account for the N-glycosylase activity of this enzyme.

Highlights

Takentogether, ourdata Previous discussionsof the substratespecificity of this enzyme suggest that the C8 keto group of 8-oxodeoxyguanine emphasized the open imidazole ring, a structural feature comand the carbonyl moiety of formamidopyrimidine en- mon to thesemodified purines andmonoadducts (Boiteuxet al., able Fpg protein to recognize and bind duplexDNA con- 1990b)
The goal of the present study was to estabstrates containing a natural abasic site were prepared by depurination of oligodeoxynucleotidescontaining a single guanine residue.‘ Oligodeoxynucleotides containing ring-opened adducts of C8-aminofluorenelish for Fpg protein quantitativbeiological/biochemical proper- guanine (ROP-3Y were producedby post-synthetic modification proceties that could be used in conjunction with structural and en- dures (Shibutani et al, 1990).All modified oligodeoxynucleotideswere ergetic data to interpret the structure-function relationsohf ipspurified by HPLC (Shibutani et al, 1990);several were purified further this DNA repair enzyme.Duplex oligodeoxynucleotide sub- by 20% PAGE (Tchou et al, 1991)
We propose a model in which Fpg protein binds to the C8-keto group of damaged purines in the major groove of duplex DNA

Summary

Introduction

The8-oxoguanine-DNAglycosylase of Escherichia DNA substrates preparedby alkylation of polynucleotides folcoli, known as formamidopyrimidine-DNA glycosy- lowed by treatment with alkali to open the purine imidazole lase (Fpg protein), has N-glycosylase and AP-lyase ac- ring (Chetsanga and Lindahl, 1979). Takentogether, ourdata Previous discussionsof the substratespecificity of this enzyme suggest that the C8 keto group of 8-oxodeoxyguanine emphasized the open imidazole ring, a structural feature comand the carbonyl moiety of formamidopyrimidine en- mon to thesemodified purines andmonoadducts (Boiteuxet al., able Fpg protein to recognize and bind duplexDNA con- 1990b). Purines containinagn intact imidazole ring were reported notto be substrates for Fpg protein (Boiteux et al, 1990b)

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Conclusion