Regulation of double-stranded DNA gap repair by the RAD6 pathway.
The RAD6 pathway allows replication across DNA lesions by either an error-prone or error-free mode. Error-prone replication involves translesion polymerases and requires monoubiquitylation at lysine (K) 164 of PCNA by the Rad6 and Rad18 enzymes. By contrast, the error-free bypass is triggered by modification of PCNA by K63-linked polyubiquitin chains, a reaction that requires in addition to Rad6 and Rad18 the enzymes Rad5 and Ubc13-Mms2. Here, we show that the RAD6 pathway is also critical for controlling repair pathways that act on DNA double-strand breaks. By using gapped plasmids as substrates, we found that repair in wild-type cells proceeds almost exclusively by homology-dependent repair (HDR) using chromosomal DNA as a template, whereas non-homologous end-joining (NHEJ) is suppressed. In contrast, in cells deficient in PCNA polyubiquitylation, plasmid repair occurs largely by NHEJ. Mutant cells that are completely deficient in PCNA ubiquitylation, repair plasmids by HDR similar to wild-type cells. These findings are consistent with a model in which unmodified PCNA supports HDR, whereas PCNA monoubiquitylation diverts repair to NHEJ, which is suppressed by PCNA polyubiquitylation. More generally, our data suggest that the balance between HDR and NHEJ pathways is crucially controlled by genes of the RAD6 pathway through modifications of PCNA.
Moertl
Simone
S
Institute for Radiobiology, Helmholtz Centre Munich-German Research Centre for Environmental Health, Ingolstaedter Landstrasse 1, Neuherberg, Germany. moertl@helmholtz-muenchen.de
Karras
Georgios I
GI
Wismüller
Tobias
T
Ahne
Fred
F
Eckardt-Schupp
Friederike
F
D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004307:Dose-Response Relationship, Radiation; D005838:Genotype; D008954:Models, Biological; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics* / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes