Bulky Lesion Bypass Requires Dpo4 Binding in Distinct Conformations

Pramodha S Liyanage,David Rueda,Louis J Romano,Alice R Walker,Alfonso Brenlla,G Andrés Cisneros

doi:10.1038/s41598-017-17643-0

Abstract

Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N2-dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.

Highlights

Mutations in genomic DNA are caused by various environmental factors, such as radiation or certain chemicals and their metabolites[1]
In high fidelity DNA polymerases, correct dNTP incorporation is accomplished by a large conformational change in the thumb and fingers domain[26], while crystal structures show that most Y-family polymerases, such as Dpo[4], do not exhibit large conformational rearrangements in the ternary complex, which is thought to affect the accuracy of nucleotide incorporation[17,27]
One means for understanding the mutagenic outcome of DNA damage is to visualize how DNA polymerases interact with lesions formed by various carcinogenic DNA adducts

Summary

Introduction

Mutations in genomic DNA are caused by various environmental factors, such as radiation or certain chemicals and their metabolites[1]. The primary consequences of these modifications are to increase B[a]P solubility and to facilitate excretion[10] During these processes, B[a]P is converted to benzo[a]pyrene diol epoxide (BPDE) which reacts with N2 of deoxyguanine (dG) to yield B[a]P-N2-dG adducts in DNA, one of which being the (+)-cis-B[a]P-N2-dG isomer (Fig. 1a)[7]. TLS is essential for cell survival, and is performed by a specialized group of DNA polymerases called Y-family polymerases, which have been found to be present in all three domains of life[15]. Extensive crystallographic studies have revealed that Dpo[4] contains a spacious active site able to accommodate two templating bases simultaneously[17] Because it has unusually small finger and thumb domains it has a very open active site with few DNA contacts leading to DNA slippage during synthesis and resulting in high levels of mismatch and frame-shift mutations[24,25]. Once translesion synthesis has occurred, the bulky adduct can distort the DNA, activating the nucleotide excision repair (NER) pathway[38,39]

Methods

Results

Conclusion