Document Detail

Assessing the requirements for nucleotide excision repair proteins of Saccharomyces cerevisiae in an in vitro system.
MedLine Citation:
PMID:  8910442     Owner:  NLM     Status:  MEDLINE    
Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiae and human cells remove the DNA lesions caused by ultraviolet light and other mutagens. This complex process involves the coordinated actions of more than 20 polypeptides. To facilitate biochemical studies of NER in yeast, we have established a simple protocol for preparing whole cell extracts which perform NER in vitro. As expected, this assay of in vitro repair was dependent on the products of RAD genes such as RAD14, RAD4, and RAD2. Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription. Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodepletion experiments to be required for NER in our yeast cell extracts. Moreover, yeast cells with temperature-sensitive mutations in the RFA2 gene, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro. These data indicate that Rpa is an essential component of the NER machinery in S. cerevisiae as it is in mammalian cells.
Z He; J M Wong; H S Maniar; S J Brill; C J Ingles
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  271     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1996 Nov 
Date Detail:
Created Date:  1996-12-30     Completed Date:  1996-12-30     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  28243-9     Citation Subset:  IM    
Banting and Best Department of Medical Research, University of Toronto, Toronto, M5G 1L6 Canada.
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MeSH Terms
Adenosine Triphosphatases*
DNA Repair*
DNA-Binding Proteins / metabolism*
Electrophoresis, Agar Gel
Fungal Proteins / metabolism*
Glycosyltransferases / metabolism*
Molecular Weight
Recombinant Proteins / metabolism
Replication Protein A
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins*
Transcription Factors*
Grant Support
Reg. No./Substance:
0/DNA-Binding Proteins; 0/Fungal Proteins; 0/RAD23 protein, S cerevisiae; 0/RAD7 protein, S cerevisiae; 0/RFA2 protein, S cerevisiae; 0/Recombinant Proteins; 0/Replication Protein A; 0/Saccharomyces cerevisiae Proteins; 0/Transcription Factors; EC 2.4.-/Glycosyltransferases; EC 3.6.1.-/Adenosine Triphosphatases; EC protein, S cerevisiae

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