Different Effects on Human Topoisomerase I by Minor Groove and Intercalated Deoxyguanosine Adducts Derived from Two Polycyclic Aromatic Hydrocarbon Diol Epoxides at or Near a Normal Cleavage Site

Yves Pommier,Glenda Kohlhagen,Gary S Laco,Heiko Kroth,Jane M Sayer,Donald M Jerina

doi:10.1074/jbc.m200209200

Abstract

Topoisomerase I (top1) relieves supercoiling in DNA by forming transient covalent cleavage complexes. These cleavage complexes can accumulate in the presence of damaged DNA or anticancer drugs that either intercalate or lie in the minor groove. Recently we reported that covalent diol epoxide (DE) adducts of benzo[a]pyrene (BaP) at the exocyclic amino group of G(+1) block cleavage at a preferred cleavage site ( approximately CTT-G(+1)G(+2)A approximately ) and cause accumulation of cleavage products at remote sites. In the present study, we have found that the 10S G(+2) adduct of BaP DE, which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation. In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(+2), which intercalate from the minor groove either between G(+1)/G(+2) or between G(+2)/A, allow normal cleavage but block religation. Both intercalated BcPh DE adducts at G(+1) suppress normal cleavage, as do both groove bound BaP DE adducts at this position. These studies demonstrate that these DE adducts provide a novel set of tools to study DNA topoisomerases and emphasize the importance of contacts between the minor groove and top1's catalytic site.

Highlights

From the ‡Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, and the ¶Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
We have found that the 10S G(؉2) adduct of BaP diol epoxide (DE), which lies toward the scissile bond in the minor groove, blocks normal cleavage, whereas the 10R isomer, which orients away from this bond, allows normal cleavage but blocks religation
In contrast to BaP, the pair of benzo[c] phenanthrene (BcPh) DE adducts at G(؉2), which intercalate from the minor groove either between G(؉1)/ G(؉2) or between G(؉2)/A, allow normal cleavage but block religation

Summary

Introduction

From the ‡Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, and the ¶Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892. We have recently utilized covalent DNA adducts (Fig. 1A) derived from trans ring opening of benzo[a]pyrene 7,8-diol9,10-epoxides (BaP DE, two enantiomers of the diastereomer in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans) by the exocyclic amino groups of the purine bases as probes of the catalytic activity of top1 [5, 6] These adducts were introduced into a 22-mer DNA sequence, which contains a single high affinity [7] top cleavage site (between T and X(ϩ1) in ϳCTT-X(ϩ1)G(ϩ2)Aϳ), where X is either G or A, Fig. 1B) and is derived from the oligonucleotide used for determination of the crystal structure of human top bound to this DNA substrate [8]. These adducts initially appear invisible to the enzyme in that normal cleavage occurs but religation is blocked [6]

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