TY - JOUR
T1 - The N-degron protein degradation strategy for investigating the function of essential genes
T2 - requirement for replication protein A and proliferating cell nuclear antigen proteins for nucleotide excision repair in yeast extracts
AU - Huang, Wenya
AU - Feaver, William J.
AU - Tomkinson, Alan E.
AU - Friedberg, Errol C.
N1 - Funding Information:
A notable feature of this study was our inability to demonstrate defective NER in vitro using CFE from a variety of existing UV radiation-sensitive rfa1 and pcna conditional mutant strains, despite the fact that these strains all carried unique mutations. We presume that the residual function of the RPA1 and PCNA proteins in these mutants is sufficient to support NER and conclude that the varying modest levels of UV radiation sensitivity they manifested reflect defects in other cellular responses to DNA damage. To circumvent the limitation of lethality associated with complete deletion or extensive disruption of the RFA1 and PCNA genes we constructed temperature-degradable strains using the strategy reported by Dohmen et al. [8] . This strategy allowed us to generate mutants in which uniquely the RPA1 or PCNA proteins were progressively inactivated in cells exposed to elevated temperature. Subsequent immediate assay of NER activity in vitro provided direct evidence that these proteins are in fact required for NER in yeast. This evidence is supported by the demonstration of partial correction by supplementing extracts with the appropriate purified protein, and by immunoneutralization experiments using specific antisera. Since the NER assay utilized measures repair synthesis, we conclude that both RPA1 and PCNA proteins are required for this event and/or any preceding events in NER. This is consistent with the results of previous studies in a human cell-free system [3, 19] .
PY - 1998/9/11
Y1 - 1998/9/11
N2 - 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). Copyright (C) 1998 Elsevier Science B.V.
AB - 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). Copyright (C) 1998 Elsevier Science B.V.
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U2 - 10.1016/S0921-8777(98)00031-7
DO - 10.1016/S0921-8777(98)00031-7
M3 - Article
C2 - 9806417
AN - SCOPUS:0031720580
VL - 408
SP - 183
EP - 194
JO - Mutation Research - DNA Repair
JF - Mutation Research - DNA Repair
SN - 0921-8777
IS - 3
ER -