Structure of a High Fidelity DNA Polymerase Bound to a Benzo[a]pyrene Adduct That Blocks Replication

Natalia P. Luneva,Lorena S. Beese,Nicholas E. Geacintov,Gerald W. Hsu,Xuanwei Huang

doi:10.1074/jbc.m411276200

Natalia P. Luneva, Lorena S. Beese + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m411276200

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Abstract

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Highlights

Benzo[a]pyrene (BP) is one of the most ubiquitous
BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases
We present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2guanine adduct base-paired with cytosine

Summary

EXPERIMENTAL PROCEDURES

Preparation of BP-modified DNA Duplexes—A Biosearch Cyclone automated DNA synthesizer was used to synthesize the oligonucleotides for this study. The oligonucleotides containing the four different stereoisomeric anti-BPN2-dG adducts were separated by reverse-phase HPLC on a 4.6 ϫ 250-mm C18 Microsorb-MV-89 column and a 13–25% 1:1 acetonitrile: methanol/triethylamine acetate (20 mM, pH 7.0) gradient. Nucleotide incorporation and extension was attempted in the crystal by transferring a crystal into stabilization solution composed of 60% saturated ammonium sulfate, 2.5% (v/v) 2-methyl-2,4-pentanediol, 100 mM MES buffer, pH 5.8, 60 mM MgSO4, and 30 mM dNTP for 24 h, 1 week, or 1 month at room temperature. A model of BP at the insertion site of BF in a ternary complex with DNA duplex and an incoming dNTP was constructed by superimposing the structure of BF bound to C:[BP]dG (primer-template) at the post-insertion site (BP2, PDB entry 1XC9) with the structure of BF bound to unmodified duplex DNA and an incoming dNTP (PDB entry 1LV5). All superpositions were done using the C␣ atoms of BF residues 646 – 655, 823– 838, and 863– 869

RESULTS

TABLE I

Position of lesion

DISCUSSION

Local base pair parametersa

Findings

Base pair step parametersa