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Preferential binding of yeast Rad4.Rad23 complex to damaged DNA
L E Jansen1, R A Verhage, J Brouwer
1MGC Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.
The Journal of Biological Chemistry
|December 5, 1998
Summary
The yeast Rad4.Rad23 protein complex is crucial for nucleotide excision repair (NER). This study shows the complex directly binds damaged DNA, suggesting a role in DNA damage recognition during NER.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Rad4 and Rad23 proteins in yeast form a complex implicated in nucleotide excision repair (NER).
- Previous genetic data suggest their involvement in early stages of NER, but their precise function remains unclear.
Purpose of the Study:
- To purify and characterize the Rad4.Rad23 complex from yeast.
- To investigate the functional role of the Rad4.Rad23 complex in nucleotide excision repair (NER).
Main Methods:
- Purification of an epitope-tagged Rad4.Rad23 complex using affinity chromatography.
- In vitro complementation assays using rad4 and rad23 mutant yeast extracts.
- Electrophoretic mobility shift assays (EMSAs) to assess DNA binding.
Main Results:
- The purified Rad4.Rad23 complex demonstrated functional activity in vitro, complementing repair defects in mutant extracts.
- The complex showed preferential binding to damaged DNA, including UV-irradiated and chemically modified DNA.
- These findings indicate the Rad4.Rad23 complex's direct interaction with DNA damage.
Conclusions:
- The yeast Rad4.Rad23 complex plays a functional role in nucleotide excision repair (NER).
- The complex appears to be involved in the recognition of DNA damage during the NER pathway.