A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro.
Nucleotide excision repair (NER) and RNA polymerase II transcription are cellular processes that require the transcription/NER factor TFIIH. We have developed a whole cell extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates. NER activity in the yeast whole cell extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein. The repair of N-acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant extracts was complemented by mixing different mutant extracts or by purified Rad proteins. Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole cell extracts. Transcription activity from the template pCYC1G- was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively. An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole cell extracts.
Wang
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Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Wu
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Friedberg
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EC
D015073:2-Acetylaminofluorene; D002474:Cell-Free System; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases; D005656:Fungal Proteins / Q000096:biosynthesis; D007700:Kinetics; D009153:Mutagens; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C073998:RAD2 protein, S cerevisiae; D051758:Transcription Factor TFIIH; D015073:2-Acetylaminofluorene; D012319:RNA Polymerase II; D004706:Endodeoxyribonucleases; D004265:DNA Helicases