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Updated: Jun 1, 2025
Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair
Published on: May 24, 2017
Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon
Ang Cai1, Katelyn L LaVigne2, Alicia M Crisalli1
1Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States.
Abstract:
Nucleotide excision repair is a crucial cellular mechanism that ensures genomic stability, thereby preventing mutations that can lead to cancer. The human XPC and its yeast ortholog Rad4 protein complexes are central to this process and were the focus of the study. We used surface plasmon resonance and differential scanning fluorimetry to study the binding characteristics of XPC and Rad4 when bound to the bulky cluster di-FAAF-containing 55-mer duplex DNA. Our findings revealed that XPC binds 10 times more significant affinity to control and di-FAAF-modified DNA than Rad4 with greater protein-DNA interactions. Differential scanning fluorimetry indicates that Rad4 causes comparatively more significant conformational changes upon complexation with the damaged DNA. We conducted DNase I footprinting of the Rad4/DNA complex for the first time by determining the regions protected from DNase I digestion. The DNA at the lesion is entirely resistant to digestion by DNase I in the absence of Rad4 several nucleotides to the 3'-side of the first FAAF lesion. The lack of DNase I cleavage at the lesions did not change upon adding Rad4. However, in the presence of Rad4, a footprint is observed on the 7-nucleotide region (5'-TGGTGAT-3') of the complementary strand to the 3' side of the lesion.
Insights
Human XPC protein shows higher affinity for damaged DNA than yeast Rad4, crucial for genomic stability and cancer prevention. This study reveals key differences in their DNA binding and conformational changes during repair.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Nucleotide excision repair (NER) is vital for maintaining genomic stability.
- The XPC protein complex (human) and Rad4 (yeast) are key players in DNA damage recognition.
- Genomic instability can lead to mutations and cancer development.
Purpose of the Study:
- To investigate and compare the DNA binding characteristics of human XPC and yeast Rad4.
- To elucidate the protein-DNA interactions with bulky, lesion-containing DNA.
- To understand the conformational changes induced by these proteins upon binding to damaged DNA.
Main Methods:
- Surface Plasmon Resonance (SPR) for binding affinity measurements.
- Differential Scanning Fluorimetry (DSF) for assessing protein-DNA complex stability and conformational changes.
- DNase I footprinting to map protein-DNA interaction sites.
Main Results:
- Human XPC exhibits a 10-fold higher binding affinity to damaged DNA compared to yeast Rad4.
- XPC demonstrates stronger overall protein-DNA interactions than Rad4.
- Rad4 induces more significant conformational changes in DNA upon binding, and its interaction site was mapped for the first time.
Conclusions:
- XPC and Rad4 display distinct binding affinities and interaction mechanisms with damaged DNA.
- These differences may reflect evolutionary adaptations in DNA repair pathways.
- The study provides novel insights into the structural basis of DNA damage recognition by NER proteins.
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