| Abstrakt: |
Nucleotide excision repair (NER) is the primary mechanism by which both Saccharomyces cerevisiaeand 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 vitrorepair was dependent on the products of RADgenes such as RAD14, RAD4,and RAD2. Interestingly, it was also dependent upon proteins encoded by the RAD7, RAD16, and RAD23genes whose precise roles in NER are uncertain, but not the RAD26gene 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 RFA2gene, 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. cerevisiaeas it is in mammalian cells. |
