Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase, Dpo4

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Bulky DNA damage poses a significant challenge to DNA replication fidelity.
• Error-prone bypass polymerases are crucial for replicating damaged DNA, but their mechanisms remain incompletely understood.
• The influence of DNA sequence context on adduct-induced mutagenesis is largely unknown.

Purpose of the Study:

• To investigate the binding conformations and DNA synthesis activity of the bypass polymerase Dpo4.
• To elucidate the distinct mechanisms by which 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) adducts affect polymerase function.
• To understand how DNA adducts influence polymerase interactions and misincorporation pathways.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) analysis to monitor polymerase-DNA complex conformations.
• DNA synthesis extension assays to evaluate polymerase activity in the presence of modified DNA templates.
• Characterization of Dpo4 interactions with DNA templates containing carcinogenic AF and AAF adducts.

Main Results:

• Both AF and AAF adducts altered Dpo4 binding in the absence of deoxynucleotide triphosphates (dNTPs).
• Addition of dNTPs led to a ternary complex with a conformation similar to that with unmodified DNA.
• Distinct misincorporation pathways were observed: AF adducts favored a primer-template loop structure, while AAF adducts utilized a dNTP-stabilized misalignment mechanism.

Conclusions:

• Dpo4 exhibits distinct conformational responses to different bulky DNA adducts.
• The presence of dNTPs can stabilize a functional polymerase-DNA complex, irrespective of the adduct type.
• The specific chemical modification of DNA adducts dictates the mechanistic pathway of polymerase misincorporation and bypass.