The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity.
The RAD6 gene of the yeast Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, DNA damage-induced mutagenesis, and sporulation. Here we demonstrate that the protein encoded by the RAD6 gene, previously shown to be a ubiquitin-conjugating (E2) enzyme, multiply ubiquitinates histones H2A and H2B efficiently to give products containing as many as seven or more molecules of ubiquitin. We also show that the highly acidic 23-residue RAD6 carboxy-terminal tail domain, which contains a total of 20 acidic residues, is essential for the histone-polyubiquitinating activity. Because the RAD6 polyacidic tail is required for the sporulation function but not for the DNA repair and induced mutagenesis functions of RAD6, the present observations suggest that the histone-polyubiquitinating activity of RAD6 protein is essential for sporulation but not for DNA repair and induced mutagenesis. Attachment of multiple molecules of ubiquitin to histones by RAD6 protein may serve to target the histones for degradation via the ubiquitin-dependent proteolytic system or to alter chromatin structure. The in vitro system for synthesizing polyubiquitinated histones described herein provides a means for investigating these possibilities.
Sung
P
P
Department of Biology, University of Rochester, New York 14627.
Prakash
S
S
Prakash
L
L
D000143:Acids / Q000502:physiology; D003001:Cloning, Molecular; D004260:DNA Repair; D005656:Fungal Proteins / Q000302:isolation & purification / Q000502:physiology*; D006657:Histones / Q000378:metabolism*; D008025:Ligases / Q000302:isolation & purification / Q000502:physiology; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D013172:Spores, Fungal; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D000143:Acids; D005656:Fungal Proteins; D006657:Histones; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases