Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair...

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• DNA repair proteins, such as Rad4/XPC, are essential for recognizing and repairing DNA damage.
• The ability of these proteins to sense damage is linked to altered DNA dynamics at lesion sites.

Purpose of the Study:

• To investigate the intrinsic dynamics of DNA containing mismatches recognized by Rad4 (yeast ortholog of XPC).
• To understand how DNA sequence and structure influence dynamics relevant to damage sensing by DNA repair proteins.

Main Methods:

• Utilized laser temperature-jump (T-jump) spectroscopy with cytosine-analog Förster Resonance Energy Transfer (FRET) probes.
• Measured intrinsic DNA dynamics in vitro for matched and mismatched DNA sequences, including AT-rich sites.
• Performed rigorous comparison with equilibrium FRET measurements.

Main Results:

• Uncovered DNA conformational dynamics across multiple timescales, differing between Rad4-specific and non-specific DNA sites.
• AT-rich non-specific sites showed dynamics within the T-jump window, with some fast kinetics (<20 μs).
• Specific sites (CCC/CCC, TTT/TTT) exhibited significantly larger fast-kinetics amplitudes and unique slow kinetics (>50 ms) at elevated temperatures, indicative of unwound/bent conformations.

Conclusions:

• Intrinsic DNA dynamics are sequence- and structure-dependent, influencing DNA deformability.
• Rapid DNA fluctuations (μs-ms) may help DNA repair proteins like Rad4 stall at damaged sites.
• Slower dynamics (>50 ms) in specific DNA suggest a propensity for conformations that facilitate Rad4 binding and damage recognition.