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Reconstitution of human DNA repair excision nuclease in a highly defined system
1Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599-7260.
Abstract:
Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (excinuclease) activity. Using the reconstituted excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.
Insights
Xeroderma pigmentosum involves faulty DNA repair. Researchers purified proteins, reconstituted DNA repair enzymes, and found accessory proteins are needed to release damaged DNA fragments.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Xeroderma pigmentosum is a hereditary DNA repair disorder.
- At least 16 polypeptides are implicated in DNA excision repair.
Purpose of the Study:
- To determine if purified proteins are sufficient for DNA excision repair.
- To investigate the mechanism of DNA lesion removal.
Main Methods:
- Purification of 16 polypeptides into five fractions.
- Reconstitution of DNA repair activity (excinuclease) using purified fractions.
- Analysis of excised DNA fragment association with protein complexes.
Main Results:
- The mixture of five purified protein fractions reconstituted excinuclease activity.
- Excised DNA fragments remained associated with the post-incision DNA-protein complex.
- This suggests accessory proteins are required for releasing the excised fragment.
Conclusions:
- The purified protein fractions are necessary but not sufficient for complete DNA excision repair.
- Accessory proteins play a crucial role in the release of DNA fragments during repair.
- Further research is needed to identify and characterize these accessory proteins.