RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO.
The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.
Hoege
Carsten
C
Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany.
Pfander
Boris
B
Moldovan
George-Lucian
GL
Pyrowolakis
George
G
Jentsch
Stefan
S
D002453:Cell Cycle; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D004247:DNA; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9