Incorporation of Nucleoside Probes Opposite O6‐Methylguanine by Sulfolobus solfataricus DNA Polymerase Dpo4: Importance of Hydrogen Bonding. Issue 13 (19th August 2013)

• Record Type: Journal Article
• Authors: Stornetta, Alessia; Angelov, Todor; Guengerich, F. Peter; Sturla, Shana J.
• Is Part Of: Chembiochem. Volume 14:Issue 13(2013)
• Page Start: 1634
• Page End: 1639
• Publication Date: 2013-08-19
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201300296
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p> <italic>O</italic> <sup>6</sup>‐Methylguanine (<italic>O</italic><sup>6</sup>‐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 <italic>O</italic><sup>6</sup>‐MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite <italic>O</italic><sup>6</sup>‐MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite <italic>O</italic><sup>6</sup>‐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 <italic>O</italic><sup>6</sup>‐MeG by Dpo4. In single‐dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to <italic>O</italic><sup>6</sup>‐MeG. Moreover, pyrimidine‐based dNTPs were generally better incorporated than purine‐based <italic>syn</italic>‐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 <italic>O</italic><sup>6</sup>‐MeG without a significant loss of efficiency.</p> </abstract>