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Role of the conserved carboxy‐terminal α‐helix of Rad6p in ubiquitination and DNA repair
Author(s) -
Dor Yuval,
Raboy Bilha,
Kulka Richard G.
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1996.671433.x
Subject(s) - biology , ubiquitin , saccharomyces cerevisiae , dna repair , biochemistry , dna , microbiology and biotechnology , ubiquitin conjugating enzyme , conserved sequence , mutant , dna ligase , ubiquitin ligase , yeast , genetics , gene , base sequence
SummaryRAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin‐conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C‐terminal α‐helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin‐conjugating enzymes (Ubcs). This specific conservation suggests that the C‐terminal a‐helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error‐free DNA repair in vivo. C‐terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C‐terminal u‐helix is also essential for error‐free DNA repair in vivo. Overexpression of Rad18p, a single‐stranded DNA‐binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C‐terminal α‐helix mutants to different degrees. This indicates that the C‐terminal α‐helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild‐type capacity for error‐free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.