Miscoding events during DNA synthesis past the nitration-damaged base 8-nitroguanine.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• 8-Nitro-2 -deoxyguanosine (8-NO(2)-dG) DNA adducts are formed by reactive nitrogen species.
• These adducts are implicated in the development of cancer within inflammatory tissues.

Purpose of the Study:

• To investigate the miscoding potential of the 8-NO(2)-dG DNA adduct.
• To determine how different mammalian DNA polymerases (pol) handle this lesion during primer extension.

Main Methods:

• Photochemical synthesis of an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct.
• Primer extension assays using various DNA polymerases (pol alpha, beta, eta, kappaDeltaC).
• Steady-state kinetic studies to analyze polymerase fidelity and miscoding frequencies.

Main Results:

• Pol alpha and beta were significantly hindered by the 8-NO(2)-dG lesion, with preferential incorporation of the correct base (dCMP).
• Pol eta and kappaDeltaC readily bypassed the lesion, exhibiting broader miscoding spectra including incorporations and deletions.
• Miscoding frequencies with pol eta and kappaDeltaC were at least 8-fold higher than with pol alpha or beta.

Conclusions:

• The miscoding potential and specificity of the 8-NO(2)-dG adduct are highly dependent on the DNA polymerase involved.
• Differential polymerase fidelity in repairing 8-NO(2)-dG adducts contributes to mutation generation.
• 8-NO(2)-dG adducts may play a crucial role in initiating inflammation-driven carcinogenesis.