Base flipping of the thymine dimer in duplex DNA

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• UV radiation exposure can cause DNA damage, forming cyclobutane pyrimidine dimers (CPDs).
• The intrahelical structure of CPD lesions is known, but the extrahelical, flipped-out conformation, essential for repair enzyme binding, remains uncharacterized.
• DNA photolyase is the enzyme responsible for CPD repair, recognizing the lesion in a specific conformation.

Purpose of the Study:

• To determine the structure of both intrahelical (flipped-in) and extrahelical (flipped-out) CPD lesions in duplex DNA.
• To calculate the potential of mean force (PMF) for the base-flipping process of CPD lesions.
• To investigate the energetic barrier for CPD base flipping and its implications for DNA repair enzyme recognition.

Main Methods:

• Molecular dynamics (MD) simulations of 8 ns were performed to model CPD lesion structures.
• Novel methodology using a two-dimensional pseudodihedral coordinate was employed to calculate the PMF.
• Free energy calculations were conducted to assess the stability of flipped-in versus flipped-out states.

Main Results:

• The structures of both flipped-in and flipped-out CPD lesions in duplex DNA were successfully calculated.
• The free energy of the flipped-out CPD state is approximately 6.5 kcal/mol higher than the flipped-in state.
• The energy barrier for flipping out is significantly lower for CPD lesions compared to undamaged DNA.

Conclusions:

• The study provides the first reported structures of the flipped-out CPD lesion.
• A lower energy barrier for flipping out suggests CPDs are predisposed to adopt a conformation recognized by DNA photolyase.
• This finding supports the hypothesis that DNA photolyase recognizes CPD lesions in their extrahelical, flipped-out state.