Incorporation of Nucleoside Probes Opposite O⁶‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding

Abstract

O6-Methylguanine (O6-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favoured opposite O6-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O6-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O6-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O6-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G relative to O6-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O6-MeG without a significant loss of efficiency

O6-Alkylguanine Postlesion DNA Synthesis Is Correct with the Right Complement of Hydrogen Bonding

“…Nucleoside analogs have been used to interrogate TLS and elucidate chemical interactions that impact DNA replication on damaged DNA substrates. − For O 6 -alkylguanine adducts, the use of synthetic nucleoside triphosphates in a TLS study showed how hydrogen bonding is important for the fidelity observed by Dpo4 to bypass O 6 -MeG . Further, nucleoside analogs with aromatic hydrocarbon functionality can stabilize duplexes containing O 6 -alkylguanine DNA adducts through base stacking interactions. , Recently, we communicated that the rates and fidelities of Dpo4-mediated synthesis past terminal base pairs containing O 6 -BnG placed opposite of nucleoside analogs differ depending on nucleobase size and shape for the nucleobase-surrogates BIM and Peri (Figure B).…”

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

“…The key artificial nucleotide used for this study was a heterocyclic imidic nucleotide that is inserted opposite O 6 -CMG but not G, therefore, marking the presence of O 6 -CMG. This approach builds on previous research concerning the interaction of artificial nucleotides with DNA adducts and polymerase-mediated amplification of damaged DNA. − The artificial nucleotide was selectively inserted opposite O 6 -CMG by a bypass-proficient KlenTaq mutant DNA Pol and required to efficiently extend a primer annealed to a DNA template containing O 6 -CMG, promoting DNA synthesis past O 6 -CMG. While the strategy has the potential to target any DNA sequence by employing the corresponding complementary sequence as a DNA primer, increased sensitivity is required to enable its utilization to interrogate defined genomic sequences for O 6 -CMG formation and persistence in biological samples to relate with CRC mutation landscapes.…”

DNA Adduct-Directed Synthetic Nucleosides

“…TLS is characterized by a nucleotide insertion and subsequent extension step to replicate past DNA damage. To understand chemical factors impacting nucleotide selection for insertion opposite O 6 -MeG, we tested the ability of the archaeal DNA polymerase Dpo4 to insert nucleoside analogues opposite O 6 -MeG . We found that synthetic dNTPs with a wide range of size and hydrogen bonding capacities were generally inserted with a higher frequency opposite G than O 6 -MeG.…”