Comparison of the in Vitro Replication of the 7-(2-Oxoheptyl)-1,N²-etheno-2′-deoxyguanosine and 1,N²-Etheno-2′-deoxyguanosine Lesions by Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4)

Abstract

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories and the results were compared to that of the 1,N2-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in a 5′-TXG-3′ local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5′-CXG-3′ local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N2-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5′-TXG-3′ sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pKa for this transition was lower than that observed for the 1,N2-ε-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pKa value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N2-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.

Replication of the 2,6-Diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (MeFapy-dGuo) Adduct by Eukaryotic DNA Polymerases

“…To better understand the mechanism of translesion synthesis (TLS), the full-length extension products were sequenced using an LC-ESI-MS-MS method previously reported (Figure S4 of the Supporting Information). ,− Briefly, a dUrd is positioned near the template–primer junction of a 5′-biotinylated primer strand. After extension with a DNA polymerase and all four dNTP’s, the extension products are immobilized on streptavidin-coated beads, which allows them to be separated from the reagents.…”

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

“…8-oxo-deoxyguanisine (8-oxo-dG) modified oligonucleotide was purchased from Midland Certified Reagent Co., tetrahydrofuran (THF) modified oligonucleotide was purchased from IDT. 12-mer oligonucleotides containing the 1, N 2 -ε-guanine [31], 7-(2-oxoheptyl)-ε-guanine [32], 8-hydroxy-1, N 2 -ε-guanine [33], 1, N 2 -propano-guanine [34], M 1 G [34], and methyl-formamidopyrimidine (MeFapy) [35], and modified 11-mer oligonucleotide ( 5’ GGCAG A *TGGTG 3’ ) containing the 7,12-dimethylbenz(a)anthracene-adenine (DMBA) [36] lesions were available from previous studies. 60 base pair (bp) and 42 bp substrates were constructed by ligation as described previously [37, 38] with the exception that fluorescein was incorporated on the damaged strand which allowed use of fluorescence instead of 32 P radioactivity as the probe.…”

Mass Spectrometry of Structurally Modified DNA

“…As a result, these specialized DNA polymerases have lower fidelity and are more likely to introduce errors, leading to mutations. − Mass spectrometry along with gel electrophoresis is an important tool employed in studying the mutagenic ability of structurally defined DNA adducts in vitro. − These in vitro translesion synthesis experiments are generally performed by annealing a DNA template strand containing a site specific DNA adduct to a complementary DNA strand (primer). The primer strand is annealed to the template, so that the 3′ terminus of the primer is positioned upstream from to the lesion , (Scheme ).…”

“…The methodology developed by Zang et al has been successfully used in translesion synthesis studies of several important DNA adducts including 1, N 2 -etheno guanine, , malondialdehyde-deoxyguanosine (M1-dG), , 7–8-dihydro-8-oxodeoxyguanosine, , O 6 -benzylguanine, , O 6 -methylguanine, , O 6 -[4-oxo-4-(3-pyridyl)butyl] ( O 6 -Pob-G), N 2 -alkyl guanine adducts, C8- and N 2 -deoxyguanosine adducts of the dietary mutagen 2-amino-3-methylimidazo[4,5-f]quinolone, N 6 -deoxyadenosine-BPDE adducts, 2,6-diamino-4-hydroxy- N 5 -(methyl)-formamidopyrimidine (MeFapy-dGuo), and 7-(2-oxoheptyl)-1, N 2 -etheno-2′-deoxyguanosine …”