The N-degron protein degradation strategy for investigating the function of essential genes: requirement for replication protein A and proliferating cell nuclear antigen proteins for nucleotide excision repair in yeast extracts.
Nucleotide excision repair (NER) of DNA in the yeast Saccharomyces cerevisiae and in human cells has been shown to be a biochemically complex process involving multiple gene products. In yeast, the involvement of the DNA replication accessory proteins, replication protein A (RPA1) and proliferating cell nuclear antigen (PCNA) in NER has not been demonstrated genetically. In this study we have generated temperature-degradable rfa1 and pcna mutants and show that these mutants are defective in NER in vitro under conditions that promote degradation of the RFA1 and PCNA gene products. We also demonstrate a physical interaction between RPA1 protein and subunits of the RNA polymerase II basal transcription factor IIH (TFIIH).
Huang
W
W
Department of Pathology, University of Texas, Southwestern Medical Center, Dallas 75235-9072, USA.
Feaver
W J
WJ
Tomkinson
A E
AE
Friedberg
E C
EC
D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D020043:Genes, Essential; D006801:Humans; D046911:Macromolecular Substances; D016296:Mutagenesis; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D013696:Temperature; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism; D019473:Transcription Factors, TFII; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; 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; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II