In vitro bypass of the major malondialdehyde- and base propenal-derived DNA adduct by human Y-family DNA polymerases κ, ι, and Rev1

Abstract

3-(2'-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M(1)dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M(1)dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M(1)dG → A and M(1)dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M(1)dG in vivo. Previous reports described the bypass of M(1)dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M(1)dG by the human Y-family DNA polymerases κ, ι, and Rev1. M(1)dG was incorporated into template-primers containing either dC or dT residues 5' to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M(1)dG:dC > M(1)dG:dG > M(1)dG:dT ∼ M(1)dG:dA, but neither hPol ι nor Rev1 extended M(1)dG-containing template-primers. Liquid chromatography-mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3'-GXC-5' template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M(1)dG in the 3'-GXT-5' template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M(1)dG residues in DNA and generate products that are consistent with some of the mutations induced by M(1)dG in mammalian cells.

Figure 1

Schematic representation of pathways generating M₁dG and/or N²-OPdG adducts.

Figure 2

Primer extension by hPol κ opposite dG and M₁dG. hPol κ (5 nM) was incubated in the presence of either unmodified or M₁dG-modified 18/23-mer radiolabeled substrate (50 nM) and four dNTPs (500 μM). Template sequences with M₁dG in 3′-GXC-5′ (A) or 3′-GXT-5′ (B) contexts were incubated for the indicated time points at 37 °C. The quenched (EDTA) samples were analyzed by 20% (w/v) denaturing polyacrylamide gel electrophoresis, and the amount of product formation was visualized using a phosphorimaging device. P represents the migration of the starting primer.

Figure 3

Single nucleotide insertion by hPol κ opposite dG and M₁dG. hPol κ (5 nM) was incubated in the presence of either unmodified or M₁dG-modified 18/23-mer radiolabeled substrate (50 nM) and a single dNTP (500 μM) as indicated. Template sequences with M₁dG in 3′-GXC-5′ (A) or 3′-GXT-5′ (B) contexts were incubated for the indicated time points at 37 °C. The quenched (EDTA) samples were analyzed by 20% (w/v) denaturing polyacrylamide gel electrophoresis, and the amount of product formation was visualized using a phosphorimaging device. P represents the migration of the starting primer.

Figure 4

Pre-steady-state kinetics of dCTP insertion by hPol κ opposite dG and M₁dG. Kinetic analysis was performed using 50 nM 18/23-mer ³²P-labeled substrate and 25 nM enzyme. Reactions were initiated by rapid addition of a 1 mM dCTP and 10 nM MgCl₂ solution to preincubated enzyme primer-template mix and quenched by the addition of EDTA after varying times ranging from 0.01 to 30 s.

Figure 5

Primer extension by hPol ι and Rev1 opposite dG and M₁dG. hPol ι or Rev1 (5 nM) was incubated in the presence of either unmodified or M₁dG-modified 18/23-mer ³²P-labeled substrate (50 nM) and four dNTPs (500 μM). The template containing 3′-G(M₁dG)C-5′ is shown, along with the results for hPol ι (A) and Rev 1 (B). Incubations were conducted for different times at 37 °C. The quenched (EDTA) samples were analyzed by 20% (w/v) denaturing polyacrylamide gel electrophoresis, and the amount of product formation was visualized using a phosphorimaging device. P represents the migration of the starting primer.

Figure 6

LC-MS analysis of hPol κ-catalyzed in vitro replication products on unmodified DNA. Oligonucleotides eluting at t_R = 3.2 min (A) were analyzed by mass spectrometry (B). Doubly and triply charged ions were detected for two products. The mass spectra and sequences of the doubly charged ions at m/z = 930 and 1087 are depicted in (C) and (D), respectively.

Figure 7

LC-MS analysis of hPol κ-catalyzed in vitro replication products of the bypass of M₁dG-modified DNA. The mass spectra for product ions eluting at (A) t_R = 2.0−2.3 min and (B) t_R = 2.9−3.2 min are shown for hPol κ-catalyzed bypass of M₁dG-containing template DNA with the 3′-GXC-5′ sequence. (C) The resulting products identified by CID fragmentation patterns are shown in schematic form. The mass spectra for product ions eluting at (D) t_R = 2.6−2.8 min and (E) t_R = 2.9−3.2 min are shown for hPol κ-catalyzed bypass of M₁dG-containing template DNA with the 3′-G(M₁dG)T-5′ sequence. (F) Products identified by CID fragmentation patterns are shown in schematic form.

Figure 8

M₁dG and γ-OH-PdG ring opening.