DNA polymerase action on benzo[a]pyrene-DNA adducts

Area of Science:

• Chemical Biology
• Molecular Biology
• DNA Damage and Repair

Background:

• Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon found in environmental pollutants.
• B[a]P undergoes metabolic activation to form reactive diol epoxides that can adduct DNA.
• Guanine residues are common targets for DNA adduct formation by B[a]P metabolites.

Purpose of the Study:

• To investigate the impact of B[a]P-guanine adduct stereochemistry on DNA replication.
• To compare the inhibitory effects of cis and trans B[a]P adducts on DNA polymerase activity.
• To assess the potential mutagenicity of B[a]P-guanine adducts.

Main Methods:

• Synthesis of a 16mer oligonucleotide containing a guanine residue modified with the (+)-enantiomer of B[a]P 7,8-dihydrodiol 9,10-epoxide.
• Separation and identification of cis and trans B[a]P-guanine adducts using chromatography, 32P postlabeling, and circular dichroism spectroscopy.
• Assessing DNA polymerase (Sequenase 2.0, human polymerase alpha) extension inhibition and nucleotide incorporation across from adducts using primer extension assays.

Main Results:

• Both cis and trans B[a]P-guanine adducts significantly inhibited DNA polymerase extension.
• The cis adduct caused more substantial inhibition of primer extension than the trans adduct.
• Nucleotide incorporation opposite the adducted guanine was severely inhibited by both adduct isomers, with deoxycytidylic acid incorporation being preferred but still significantly reduced.

Conclusions:

• The stereochemistry of B[a]P-guanine adducts influences their ability to block DNA replication.
• Cis adducts represent a more effective block to DNA replication compared to trans adducts.
• These findings suggest that B[a]P-guanine adducts may not be highly efficient mutagenic lesions due to replication stalling.