"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• DNA lesion recognition by repair proteins is crucial for genome stability.
• Altered DNA dynamics at damage sites are proposed to facilitate protein binding.
• Previous studies indicated DNA unwinding at lesion sites but lacked dynamic information.

Purpose of the Study:

• To investigate the dynamics and timescales of DNA conformational changes at lesion sites.
• To determine if DNA flexibility at damage sites overlaps with DNA repair protein scanning times.
• To understand the role of DNA dynamics in DNA damage sensing.

Main Methods:

• Utilized Förster Resonance Energy Transfer (FRET) with Atto550/Atto647N dyes for DNA labeling.
• Employed Fluorescence Correlation Spectroscopy (FCS) to measure DNA dynamics.
• Examined DNA oligomers with 3 base pair mismatched sites recognized by Rad4.

Main Results:

• Confirmed significant unwinding/bending deformations in mismatched DNA compared to matched DNA.
• Fluorescence Correlation Spectroscopy (FCS) revealed spontaneous DNA deformations occurring at ~300 µs.
• No significant fluctuations were detected for matched DNA within the FCS time window (600 ns–10 ms).

Conclusions:

• Demonstrated anomalous unwinding/bending fluctuations in mismatched DNA on microsecond timescales.
• These DNA dynamics overlap with the stepping times of DNA repair proteins (~<500 µs).
• Proposed that these "flexible hinge" dynamics at lesion sites can arrest diffusing proteins, facilitating damage interrogation and recognition.