Abstract
Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N2-propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.
Highlights
Acrolein, an ␣,-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant
We identify the translesion synthesis (TLS) Pols required for replicating through the (r) ␥-HOPdG adduct in human cells and show that TLS opposite this adduct is mediated via three independent pathways that involve Rev1 and Pol in one pathway, Pol and Pol in another pathway, and Pol in the third pathway, and TLS by all of these pathways is mediated in a predominantly error-free manner
Our genetic observations indicating that replication through the (r) ␥-HOPdG adduct is mediated by three independent pathways, composed of Rev1/Pol, Pol/Pol, and Pol, respectively (Fig. 2), differ strikingly from the roles predicted for TLS Pols from biochemical studies
Summary
The reaction of acrolein with the N2 of guanine in DNA followed by ring closure results in the formation of the cyclic adduct ␥-hydroxy-1,N2-propano-2Ј-deoxyguanosine (␥-HOPdG).2 ␥-HOPdG can exist in DNA in a ring-closed or ring-opened form (8 –10). ␥-HOPdG presents a strong block to synthesis by replicative DNA polymerases, and it is inhibitory to synthesis by yeast and human Pol, at the nucleotide (nt) incorporation step [11, 12]. In the presence of a reducing agent, ␥-HOPdG can be trapped as the N2-(3-hydroxypropyl) 2Ј-deoxyguanosine adduct, which permanently stays in the ring-opened configuration (Fig. 1A). Biochemical studies with (r) ␥-HOPdG have indicated that both yeast and human Pol can carry out proficient synthesis opposite this adduct by inserting the correct nt and by extending synthesis [11, 13]; Pol performs proficient TLS opposite this adduct by inserting the correct nt and by extending synthesis [13]. We identify the TLS Pols required for replicating through the (r) ␥-HOPdG adduct in human cells and show that TLS opposite this adduct is mediated via three independent pathways that involve Rev and Pol in one pathway, Pol and Pol in another pathway, and Pol in the third pathway, and TLS by all of these pathways is mediated in a predominantly error-free manner. We discuss the possible implications of these observations for TLS opposite (r) ␥-HOPdG and other minor groove DNA lesions
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