Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M₁dG, the Genomic Metabolite of M₁dG, and Liquid Chromatography–Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι

Abstract

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M 1 dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M 1 dG in genomic DNA is enzymatically oxidized to 6-oxo-M 1 dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M 1 dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M 1 dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography−tandem mass spectrometry (LC−MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M 1 dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M 1 dG blocks further replication by the enzyme.

Base-Displaced Intercalated Structure of the 3-(2-Deoxy-β-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG) Lesion in DNA

“…The complementary cytosine C 21 was extruded out of the duplex and toward the major groove, which allowed the bulky 6-oxo-M 1 dG lesion to be accommodated within the helix. The localization of the structural perturbations to the damaged base pair X 4 :C 21 and its 5′-and 3′-neighboring base pairs T 3 :A 22 and A 5 :T 20 was evident; the flanking base pairs remained within the Watson−Crick hydrogen bonding contact. Figure 10 shows views along the helical axis showing base stacking interactions.…”

“…Figure 6 summarizes chemical shift perturbations induced in the 6-oxo-M 1 dGmodified 5′-d(C 1 A 2 T 3 X 4 A 5 T 6 G 7 A 8 C 9 G 10 C 11 T 12 )-3′:5′-d-(A 13 G 14 C 15 G 16 T 17 C 18 A 19 T 20 C 21 A 22 T 23 G 24 )-3′ duplex as compared to the corresponding unmodified duplex. These were clustered at the damage site X 4 :C 21 and the two flanking base pairs 5′ and 3′ to the 6-oxo-M 1 dG lesion. The greatest perturbation was observed for the C 21 nucleobase, which was complementary to the 6-oxo-M 1 dG modified X 4 nucleotide.…”

“…These are collected in Table 1. Because the 6-oxo-M 1 dG lesion created structural perturbation at base pairs T 3 :A 22 and X 4 :C 21 , canonical restraints were not employed for X 4 , A 5 , T 20 , C 21 , and A 22 in the 6-oxo-M 1 dG modified region, nor were they employed at the terminal nucleotides C 1 , T 12 , A 13 , and G 24 . Each of the 20 emergent structures was further subjected to potential energy minimization using the conjugate gradients algorithm.…”

“…Christov et al demonstrated that hPol ι incorporated deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M 1 dG with approximately equal efficiencies, but deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) were poor substrates. Moreover, after incorporation of a single dNTP opposite the 6-oxo-M 1 dG, the lesion blocked further replication . More recent studies examined mutagenic bypass of 6-oxo-M 1 dG by additional Y-family polymerases .…”

“…When placed opposite dC, it rearranges to N 2 -oxopropenyl-dG (OpdG) . It also may be oxidized to 3-(2-deoxy-β-D-erythropentofuranosyl)-pyrimido­[1,2- f ]­purine-6,10­(3 H ,5 H )-dione (6-oxo-M 1 dG). − The Y-family polymerase hPol η preferentially inserts dAMP or dGMP across from 6-oxo-M 1 dG, suggesting that like M 1 dG, , 6-oxo-M 1 dG is genotoxic. Crystal structures of hPol η forming both insertion and extension complexes in which template-primer oligodeoxynucleotide duplexes contained a site-specific 6-oxo-M 1 dG lesion suggested that 6-oxo-M 1 dG blocks DNA replication and is mutagenic …”