A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro

被引:21
作者
Wang, ZG [1 ]
Wu, XH [1 ]
Friedberg, EC [1 ]
机构
[1] UNIV TEXAS, SW MED CTR, DEPT PATHOL, LAB MOL PATHOL, DALLAS, TX 75235 USA
来源
MUTATION RESEARCH-DNA REPAIR | 1996年 / 364卷 / 01期
关键词
DNA repair; transcription; cell-free system; yeast extract;
D O I
10.1016/0921-8777(96)00019-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 pCYClG(-) 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.
引用
收藏
页码:33 / 41
页数:9
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