ROLE OF THE CONSERVED CARBOXY-TERMINAL ALPHA-HELIX OF RAD6P IN UBIQUITINATION AND DNA-REPAIR

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjuga ting enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p requi re the ubiquitination of target proteins, but its substrates as well a s other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-termin al alpha-helix, which are conserved from yeast to man but are absent f rom most other yeast ubiquitin-conjugating enzymes (Ubcs). This specif ic conservation suggests that the C-terminal alpha-helix is important for the unique activities of the Rad6p family of Ubcs. We have investi gated the effects of mutating this highly conserved region on the ubiq uitination of model substrates in vitro and on error-free DNA repair i n vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the poss ibility that Rad6p interacts with its substrates in vivo by similar me chanisms. The distal part of the C-terminal alpha-helix is also essent ial for error-free DNA repair in vivo. Overexpression of Rad18p, a sin gle-stranded DNA-binding protein that also interacts with Rad6p, allev iates the DNA repair defects of the C-terminal alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-helix of R ad6p mediates its interaction with Rad18p, an essential step in DNA re pair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants a ffecting several functions of the 26S proteasome retain wild-type capa city for error-free DNA repair. This raises the possibility that ubiqu itination by Rad6p in DNA repair does not target proteins for proteaso mal degradation.