Volume 271, Number 45,
Issue of November 8, 1996
pp. 28243-28249
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Assessing the Requirements for Nucleotide Excision Repair
Proteins of Saccharomyces cerevisiae in an in
Vitro System
(Received for publication, March 4, 1996, and in revised form, August 20, 1996)
Zhigang
He
,
Johnson M. S.
Wong
,
Hina S.
Maniar
¶
,
Steven J.
Brill
¶
and
C. James
Ingles
From the 
Banting and Best Department of Medical
Research and Department of Molecular and Medical Genetics, University
of Toronto, Toronto, M5G 1L6 Canada and the ¶ Department of
Molecular Biology and Biochemistry, Center for Advanced
Biotechnology and Medicine, Rutgers University,
Piscataway, New Jersey 08855
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.
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