Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome.
In yeast and humans, nucleotide excision repair (NER) of ultraviolet (UV)-damaged DNA requires a large number of highly conserved protein factors, which include the multisubunit RNA polymerase II transcription factor TFIIH. Here, we examine whether NER occurs by sequential assembly of different repair factors at the site of DNA damage or by the placement there of a "preformed" repairosome containing TFIIH and all the other essential NER factors. Contrary to the recent report (Svejstrup, J. Q., Wang, Z., Feaver, W. J., Wu, X., Bushnell, D. A., Donahue, T. F., Friedberg, E. C., and Kornberg, R. D. (1995) Cell 80, 21-28), our results provide no evidence for a pre-assembled repairosome; instead, they support the sequential assembly model. By several independent criteria, including co-purification, immunoprecipitation, and gel filtration of homogeneous proteins, we show that the damage recognition factor Rad14 exists in a ternary complex with the Rad1-Rad10 nuclease. We also find that Rad14 interacts directly with Rad1, but only slightly with Rad10, and that it interacts with the Rad1-Rad10 complex much more efficiently than with Rad1 alone. In the reconstituted NER system, a higher level of incision of UV-damaged DNA is achieved with the Rad1-Rad10-Rad14 complex, which we designate as nucleotide excision repair factor-1, NEF-1.
Guzder
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Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, 77555-1061, USA.
Sung
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Prakash
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Prakash
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D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004720:Endonucleases / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D011503:Transglutaminases; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; C072975:RAD14 protein, S cerevisiae; C082463:RAD4 protein, S pombe; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D011503:Transglutaminases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C499354:rad1 protein, S pombe; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes