Position-specific Suppression and Enhancement of HIV-1 Integrase Reactions by Minor Groove Benzo[a]pyrene Diol Epoxide Deoxyguanine Adducts: IMPLICATIONS FOR MOLECULAR INTERACTIONS BETWEEN INTEGRASE AND DNA SUBSTRATES

Allison A Johnson,Jane M Sayer,Haruhiko Yagi,Govind P Kalena,Ronak Amin,Donald M Jerina,Yves Pommier

doi:10.1074/jbc.m311263200

Abstract

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.

Highlights

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication
To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA
Binding of Integrase to Modified Oligonucleotides Containing BaP DE dG Adducts—Before investigating integrase catalytic reactions, we examined the binding of integrase to the adducted oligonucleotides using the Schiff base cross-link assay between integrase and its DNA substrate [24]

Summary

Introduction

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. The exposed 3Ј-hydroxyl at the cleavage site is used by integrase for nucleophilic attack upon a target DNA, resulting in insertion of the viral genome into a target chromosome (strand transfer, ST) (Fig. 2A). Disulfide cross-linking revealed proximity of the integrase amino acid 246 when mutated from glutamic acid to cysteine and the seventh base from the 5Ј-end of the lower strand of the U5 LTR [1]. These four contacts have been useful for general alignment of

Methods

Results

Conclusion