Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Biomolecular structural changes are crucial for function, but studying these dynamics is challenging.
• Light-triggered oligonucleotide modifications offer new ways to study molecular interactions.
• The Rad4/XPC complex initiates eukaryotic nucleotide excision repair (NER) by recognizing DNA lesions.

Purpose of the Study:

• To investigate the use of photocleavable DNA to modulate Rad4/XPC DNA repair complex binding using light.
• To characterize the structural and binding interactions of NPOM-modified DNA with Rad4/XPC.

Main Methods:

• Synthesis and characterization of 6-nitropiperonyloxymethyl (NPOM)-modified DNA.
• Assessment of Rad4/XPC binding to NPOM-DNA using light-induced cleavage.
• Fluorescence lifetime-based DNA conformational analysis.
• Computational modeling including conformational searches and molecular dynamics simulations.

Main Results:

• NPOM-modified DNA is recognized by Rad4/XPC.
• Light-induced cleavage of NPOM from DNA dose-dependently abolishes Rad4/XPC binding.
• NPOM-DNA maintains a B-DNA-like conformation, but Rad4 binding induces distortion.
• Molecular simulations suggest NPOM resides in the DNA major groove with minimal perturbation of base stacking.

Conclusions:

• Photoactivable DNA, specifically NPOM-modified DNA, can be used to control Rad4/XPC binding with light.
• This system provides a novel approach to study DNA repair pathways like NER.
• NPOM-modified DNA can serve as a surrogate for DNA lesions in mechanistic studies.