Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Nucleotide excision repair (NER) is a crucial DNA repair pathway conserved across species.
• NER removes helix-destabilizing DNA lesions, including UV-induced photoproducts.
• Understanding the initial damage recognition step is vital for comprehending NER efficiency.

Purpose of the Study:

• To investigate the dynamic protein-DNA interactions during the damage recognition phase of NER.
• To elucidate the role of specific protein domains, such as the β-hairpin domain 3 (BHD3) of Rad4, in DNA lesion detection.
• To characterize the motion and binding behavior of Rad4-Rad23 complexes at DNA damage sites.

Main Methods:

• Single-molecule fluorescence microscopy was employed to observe dynamic protein-DNA interactions.
• Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) was used to track complex formation and motion.
• Atomic force microscopy was utilized to analyze the structural role of Rad4's BHD3 domain.

Main Results:

• Rad4-Rad23 complexes were observed forming non-motile complexes or performing one-dimensional searches via diffusion or constrained motion.
• The BHD3 domain of Rad4 was found to be non-essential for damage-specific binding and DNA bending.
• Deletion of specific residues in the BHD3 tip enhanced Rad4-Rad23 constrained motion but did not impair in vivo UV resistance or photoproduct repair.

Conclusions:

• A distinct intermediate in NER damage recognition allows for dynamic DNA damage detection.
• The BHD3 domain's role in constrained motion suggests a mechanism for efficient lesion scanning.
• These findings contribute to a deeper understanding of the molecular mechanisms underlying DNA repair.