Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase, Dpo4.
Bulky DNA damage inhibits DNA synthesis by replicative polymerases and often requires the action of error prone bypass polymerases. The exact mechanism governing adduct-induced mutagenesis and its dependence on the DNA sequence context remains unclear. In this work, we characterize Dpo4 binding conformations and activity with DNA templates modified with the carcinogenic DNA adducts, 2-aminofluoene (AF) or N-acetyl-2-aminofluorene (AAF), using single-molecule FRET (smFRET) analysis and DNA synthesis extension assays. We find that in the absence of dNTPs, both adducts alter polymerase binding as measured by smFRET, but the addition of dNTPs induces the formation of a ternary complex having what appears to be a conformation similar to the one observed with an unmodified DNA template. We also observe that the misincorporation pathways for each adduct present significant differences: while an AF adduct induces a structure consistent with the previously observed primer-template looped structure, its acetylated counterpart uses a different mechanism, one consistent with a dNTP-stabilized misalignment mechanism.
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
Alfonso
A
Brenlla
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA Department of Medicine, Section of Virology, Imperial College London, London, UK Single Molecule Imaging Group, MRC Clinical Sciences Centre, Imperial College London, London, UK david.rueda@imperial.ac.uk.
David
D
Rueda
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA ljr@chem.wayne.edu.
Louis J
LJ
Romano
D015073:2-Acetylaminofluorene / Q000737:chemistry*; D002273:Carcinogens / Q000737:chemistry*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000378:metabolism*; D017931:DNA Primers; D005449:Fluorenes / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D013698:Templates, Genetic
D002273:Carcinogens; D018736:DNA Adducts; D017931:DNA Primers; D005449:Fluorenes; D009711:Nucleotides; C012177:2-aminofluorene; D006147:Guanine; D004247:DNA; D015073:2-Acetylaminofluorene; D019951:DNA Polymerase beta