Base pairing and replicative processing of the formamidopyrimidine-dG DNA lesion

Area of Science:

• Molecular Biology
• DNA Damage and Repair
• Biochemistry

Background:

• Oxidative stress induces DNA lesions, including the major lesion 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2 -deoxyguanosine (FaPydG).
• Understanding the base pairing and coding potential of FaPydG is crucial for estimating its mutagenic effects.

Purpose of the Study:

• To investigate the base pairing properties and thermodynamic stability of a cyclopentane-based analogue of FaPydG (cFaPydG) within DNA duplexes.
• To determine the mutagenic potential and replication fidelity of the FaPydG lesion using kinetic primer extension studies.

Main Methods:

• Synthesis of oligonucleotides containing cFaPydG, cyclopentane analogue of 2 -deoxyguanosine (cdG), and 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG).
• Determination of duplex thermodynamic stability using concentration-dependent melting-point measurements (van't Hoff plots).
• Kinetic primer extension studies employing Saccharomyces cerevisiae Pol eta.

Main Results:

• The cFaPydG lesion significantly destabilizes DNA duplexes, with cytosine (dC) being the optimal base pairing partner.
• FaPydG is unable to form a stable base pair with adenine (dA), unlike 8-oxodG.
• Kinetic studies reveal that cFaPydG is replicated error-free by S. cerevisiae Pol eta, with efficient insertion of dC, indicating retained coding potential.

Conclusions:

• The FaPydG lesion, despite causing duplex destabilization, retains the coding potential of guanine (dG) by preferentially pairing with cytosine.
• The cyclopentane substitution in cdG has a marginal effect on duplex stability compared to canonical deoxyguanosine (dG).
• FaPydG represents a DNA lesion that can be accurately replicated, potentially mitigating its mutagenic impact.