4-1) CHARACTERIZATION AND EXPRESSION OF RAD<SUB>4</SUB> GENE INVOLVED IN NUCLEOTIDE EXCISION REPAIR OF UV-DAMAGED Saccharomyces cerevisiae

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Saccharomyces cerevisiae express RAD4 gene for nucleotide excision repair of UV-induced DNA damages. Upon complementation with rad&lt4-4&gt mutant, a 7.6kb clone containing the RAD4 gene designated as pPCl was isolated from a yeast genomic library. The pPCl was further narrowed to 2.5kb flanked with BglII and BartHI sites. The cloned RAD4 gene was found to propagate in E. coli without loss of its complementing activity. Pulse-field gel electrophoresis indicated that the cloned RAD4 gene was localized in the right arm of chromosome V. DNA-tRNA hybridization revealed that the cloned gene did not contain a suppressor tRNA gene. The rad4 mutants with various plasmids containing the cloned RAD4 gene, regardless of their copy number, had enhanced resistance against UV damages equivalent to that found in wild type. As determined by Sl nuclease digestion, the RAD4 transcript was found to be 2.3kb in size and the Sl nuclease mapping revealed the production of a protected fragment of 760 nucleotides within the transcript. Transcriptional start point was found at 48 base pairs upstream from the first ATG codon of the translation initiation codon. The overexpressed Rad4 protein was estimated to be 89kD and confirmed the expected size based on the actual length of RAD4 gene. Upon stationary phase culturing, E. coli cells transformed with the cloned RAD4 gene had a delayed enterance into exponential growth phase and produced reduced amount of host proteins. These results have indicated that the pPCl is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplication in E. coli.

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