Role of the conserved carboxy-terminal alpha-helix of Rad6p in ubiquitination and DNA repair.
RAD6 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 alpha-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 alpha-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 alpha-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 alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-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.
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Y
Y
Dor
B
B
Raboy
R G
RG
Kulka
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004260:DNA Repair; D004271:DNA, Fungal; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases