Base pair opening in a deoxynucleotide duplex containing a cis-syn thymine cyclobutane dimer lesion

Area of Science:

• Molecular Biology
• Biophysics
• Photochemistry

Background:

• The cis-syn thymine cyclobutane dimer is a major DNA photoproduct formed by UV radiation.
• This lesion is strongly implicated in the initiation of skin cancer.
• Understanding its impact on DNA structure and stability is crucial for cancer research.

Purpose of the Study:

• To quantitatively assess the thermodynamic stability of base pairs flanking a cis-syn thymine cyclobutane dimer.
• To compare the stability of individual base pairs in dimer-containing duplexes versus undamaged DNA.
• To elucidate the mechanism of DNA destabilization caused by the thymine dimer lesion.

Main Methods:

• UV melting thermodynamic measurements to determine duplex stability.
• Circular dichroism (CD) spectroscopy to analyze DNA conformation.
• 2D NOESY NMR spectroscopy to study base pairing and dynamics.
• NMR magnetization transfer to measure imino proton exchange rates and determine base pair opening equilibrium constants (K(op)).

Main Results:

• The thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex.
• Base pair opening equilibrium constants (K(op)) were determined for each base pair.
• In undamaged duplexes, K(op) increases from the center to the ends.
• In dimer-containing duplexes, base pair opening at the 5utoxyT of the dimer is facile (K(op) = 3 × 10⁻⁴), significantly destabilizing this region.
• The 3utoxyT of the dimer shows more stable base pairing than the 5utoxyT.
• The dimer asymmetrically destabilizes the duplex, with greater impact on the 5utoxy side.

Conclusions:

• The cis-syn thymine cyclobutane dimer asymmetrically destabilizes DNA duplexes, primarily by facilitating base pair opening on its 5utoxy side.
• The observed opening mechanism suggests it is not a simple flipping out of the dimer as a single unit.
• These findings provide insights into the structural consequences of UV-induced DNA damage and its potential role in mutagenesis and carcinogenesis.