Reconstitution of Yeast Nucleotide Excision Repair with Purified Rad Proteins, Replication Protein A, and Transcription Factor TFIIH (*)
- From the (1) Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
- § To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th & Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8602; Fax: 409-747-8608.
Abstract
Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5′-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.
Footnotes
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↵* This work was supported by Grants CA35035 and CA41621 from the National Cancer Institute and Grant DE-FGO3-93ER61706 from the Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- NER
-
nucleotide excision repair
- PAGE
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polyacrylamide gel electrophoresis
- RPA
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replication protein A
- ATP
S -
adenosine 5′-O-(3-thiotriphosphate)
- AAF
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N-acetoxy-2-aminoacetylfluorene.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
