Characterization of RAD4 gene required for ultraviolet-induced excision repair of Saccharomyces cerevisiae propagated in Escherichia coli without inactivation.

The previously isolated RAD4 gene designated as pPC1 from the genomic library of Saccharomyces cerevisiae (Yoon et al., 1985, Korean J. Genetics 7, 97-104) appeared to propagate in Escherichia coli and yet retained its complementing activity to rad4 mutants without inactivation. The subcloned RAD4 gene was found to be localized within a 2.5 kb DNA fragment flanking Bg1II and BamHI sites in the insert DNA, and was shown to have the same restriction map as a yeast chromosomal DNA, as determined by Southern hybridization. Tetrad analysis and pulse-field chromosome mapping have revealed that the cloned RAD4 gene can be mapped and integrated into the yeast chromosome V, the actual site of this gene. DNA-tRNA hybridization has shown that the isolated RAD4 gene did not contain a suppressor tRNA gene. These results have indicated that the pPC1 is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplification in E. coli.