Carcinogenic adducts induce distinct DNA polymerase binding orientations.

Kyle B Vrtis,David Rueda,Louis J Romano,Radoslaw P Markiewicz

doi:10.1093/nar/gkt554

Kyle B Vrtis, David Rueda + Show 2 more

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https://doi.org/10.1093/nar/gkt554

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Abstract

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism.

Highlights

Most cancers propagate from an accumulation of mutations in genes that control cell growth [1,2]
To measure the degree of inhibition by either an AF-dG or AAF-dG adduct positioned in our particular primer– template system (Supplementary Table S1), we measured running start DNA synthesis on the same AF or AAFmodified DNA template sequences used in the singlemolecule studies
Gaining an understanding of how a DNA polymerase interacts with DNA adducts formed by chemical carcinogens is an important goal because these interactions are the basis for the mutagenic effects of these DNA lesions

Summary

Introduction

Most cancers propagate from an accumulation of mutations in genes that control cell growth [1,2]. Arylamines are a well-studied class of carcinogen found in numerous occupational settings, tobacco smoke and chemical dyes [4,5,6]. AAF-dG and AF-dG adducts differ only by an acetyl group, the two adducts have different structure in duplex DNA [10,11,12,13]. Both adducts display considerable conformational heterogeneity, the major conformation of the AAF-dG in duplex DNA has the guanine rotated into a syn conformation with the AAF moiety intercalated into the DNA helix. Numerous solution structures of AF-dG adducts in several duplex sequence contexts indicate that in most sequences, the major structure is one where the AF resides in the major groove with the guanine in a normal anti orientation so that proper base paring can occur with a complementary cytosine

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