DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Abstract

The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.