Human and yeast DNA damage recognition complexes bind with high affinity DNA structures mimicking in size transcription bubble

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Biochemistry

Background:

• The XPC-RAD23B complex is crucial for initiating global genome nucleotide excision repair (GG-NER) in humans.
• Its yeast ortholog, Rad4-Rad23, plays a similar role in DNA repair pathways.
• Understanding the DNA binding specificities of these complexes is vital for elucidating DNA repair initiation.

Purpose of the Study:

• To analyze the interaction of human XPC-RAD23B and yeast Rad4-Rad23 complexes with damaged DNA.
• To quantitatively assess the binding affinities of these complexes to various DNA structures.
• To investigate the relationship between DNA structure, bending, and protein binding affinity.

Main Methods:

• Electrophoretic mobility shift assay (EMSA) to compare DNA binding of human and yeast orthologs.
• Fluorescent polarization (FP) measurements for quantitative analysis of protein-DNA interactions.
• Utilized model DNA structures, including damaged and undamaged bubbles and duplexes.

Main Results:

• Human XPC-RAD23B and yeast Rad4-Rad23 exhibit similar DNA binding behaviors.
• Both complexes show the highest binding affinity for damaged bubble-DNA structures (~10^-10 M or less).
• Binding affinity decreases in the order: damaged bubble > undamaged bubble > damaged duplex > undamaged duplex, correlating with DNA bending angle.

Conclusions:

• The affinity of XPC/Rad4 complexes for DNA is directly related to the degree of DNA structural deviation.
• Increased DNA bending correlates with higher binding affinity, highlighting the importance of DNA conformation in repair initiation.
• These findings provide detailed insights into the molecular mechanisms underlying DNA damage recognition by key NER proteins.