Premium
Incorporation of Nucleoside Probes Opposite O 6 ‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding
Author(s) -
Stornetta Alessia,
Angelov Todor,
Guengerich F. Peter,
Sturla Shana J.
Publication year - 2013
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300296
Subject(s) - dna polymerase , polymerase , sulfolobus solfataricus , dna , primer (cosmetics) , chemistry , biochemistry , stereochemistry , gene , organic chemistry , archaea
O 6 ‐Methylguanine ( O 6 ‐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 favored opposite O 6 ‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O 6 ‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O 6 ‐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 O 6 ‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O 6 ‐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 O 6 ‐MeG without a significant loss of efficiency.