Mechanism of error-free replication across benzo[a]pyrene stereoisomers by Rev1 DNA polymerase

Olga Rechkoblit,Hannah Landes,Aneel K Aggarwal,Alexander Kolbanovskiy,Nicholas E Geacintov

doi:10.1038/s41467-017-01013-5

Olga Rechkoblit, Hannah Landes + Show 3 more

Open Access

https://doi.org/10.1038/s41467-017-01013-5

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Abstract

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N2 amino group of guanine to generate four stereoisomeric BP-N2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N2-dG adducts, namely the 10S (+)-trans-BP-N2-dG, 10R (+)-cis-BP-N2-dG, and 10S ( − )-cis-BP-N2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.

Highlights

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N2 amino group of guanine to generate four stereoisomeric BP-N2-dG adducts
Pol κ and Rev[1], can efficiently and preferentially insert the correct cytosine base opposite the BP-N2-dG adducts in vitro[22,23,24,25,26], while Pol η is proficient in incorporating mutation-inducing adenine base[23, 27], and Pol ι is blocked by the adducts[23]
Site- and stereo- modified 17-mer DNA oligonucleotides with single (+)-trans-BP-N2-dG, (−)-trans-BP-N2-dG, (+)-cis-BP-N2-dG, or (−)-cis-BP-N2-dG adducts (Fig. 1b) were generated by the direct synthesis method[50] using racemic (±)-anti-BPDE

Summary

Introduction

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N2 amino group of guanine to generate four stereoisomeric BP-N2-dG adducts. The metabolic activation of BP in human cells gives rise to a pair of mirror image BP diol epoxides, the (+)-antiBPDE and (−)-anti-BPDE enantiomers (Fig. 1a), each of which reacts predominantly with the exocyclic N2 amino group of guanine by trans- and cis- epoxide ring opening. This leads to four possible stereoisomeric BP-N2-dG adducts (Fig. 1b). The B-family TLS Pol ζ, a “universal extender”, is able to elongate from either the correct or incorrect bases inserted by other polymerases (including Rev1) across the lesion site[28,29,30]

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Results

Conclusion