STABLE ESTER CONJUGATE BETWEEN THE SACCHAROMYCES-CEREVISIAE RAD6 PROTEIN AND UBIQUITIN HAS NO BIOLOGICAL-ACTIVITY

The RAD6 gene of Saccharomyces cerevisae, which encodes a ubiquitin-conjugating enzyme, is required for DNA repair, DNA damage-induced mutagenesis and sporulation. To evaluate the biological relevance of the thioester adduct between RAD6 protein and ubiquitin, formed as an obligatory, transient intermediate during ubiquitin conjugation to substrates, we 3ltered cysteine 88 in RAD6 to serine. Esterification with ubiquitin occurs at serine 88 in the mutant protein, but conjugation of ubiquitin to the test substrate histone H2A is inactivated. Phenotypically, strains harboring the rad6 Ser88 allele are indistinguishable from rad6 deletion (rad6Δ) mutant cells. These findings argue against ligation of ubiquitin at cysteine 88 acting as a functional switch of a cryptic biochemical activity in RAD6. © 1991.