Bulky Lesion Bypass Requires Dpo4 Binding in Distinct Conformations

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-N 2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2-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.

Computational Simulations of DNA Polymerases: Detailed Insights on Structure/Function/Mechanism from Native Proteins to Cancer Variants

“…We performed classical MD simulations on Dpo4 with B[a]P incorporated to the DNA in pure water and in the water/DMSO mixture. 101 Our results indicate that the B[a]P indeed intercalates with the DNA and does not allow the adduct to be everted from the double strand when in pure water. Conversely, in the water/DMSO mixture, the B[a]P adduct flips out into an open space next to the active site underneath the finger domain in a solvent exposed conformation, allowing replication to continue ( Figure 12 ).…”

“…We performed classical MD simulations on Dpo4 with B­[a]P incorporated to the DNA in pure water and in the water/DMSO mixture . Our results indicate that the B­[a]P indeed intercalates with the DNA and does not allow the adduct to be everted from the double strand when in pure water.…”

“…The experimental results indicate that the cis conformation of B[a]P adducted to guanine exhibits two different behaviors depending on the solvent environment; in a pure water system, the adduct takes the place of the guanine residue, intercalated within the DNA helix, and replication does not proceed past that point. 101 In a 10% dimethyl sulfoxide (DMSO)/90% water mixture, two different FRET states are observed and Dpo4 is able to continue the DNA synthesis (TLS). Additionally, a G-G mismatch is more stable than the correct G-C base pairing, resulting in increased errors in TLS.…”

“…We have recently carried out computational simulations to aid in the understanding of biochemical and single molecule FRET experiments for Dpo4 with B­[a]P damage. The experimental results indicate that the cis conformation of B­[a]P adducted to guanine exhibits two different behaviors depending on the solvent environment; in a pure water system, the adduct takes the place of the guanine residue, intercalated within the DNA helix, and replication does not proceed past that point . In a 10% dimethyl sulfoxide (DMSO)/90% water mixture, two different FRET states are observed and Dpo4 is able to continue the DNA synthesis (TLS).…”

Conformational Dynamics of DNA Polymerases Revealed at the Single-Molecule Level

“…In an early application, smFRET was used to monitor the movement of KF on a DNA template during DNA synthesis, enabling measurement of primer elongation with single base-pair resolution (Christian et al, 2009). This approach was also used to elucidate the impact of bulky benzopyrene adducts on translesion DNA synthesis by the Y family polymerase Dpo4 (Liyanage et al, 2017).…”