TY - JOUR
T1 - Regulation of double-stranded DNA gap repair by the RAD6 pathway
AU - Moertl, Simone
AU - Karras, Georgios I.
AU - Wismüller, Tobias
AU - Ahne, Fred
AU - Eckardt-Schupp, Friederike
N1 - Funding Information:
We thank Klaudia Winkler for excellent technical help and Helle Ulrich for the gift of plasmid YIPlac211-C914S, and Lucian Moldovan for discussion. Furthermore we would like to thank Stefan Jentsch and Boris Pfander for valuable suggestions and for carefully revising the manuscript. We also thank the Federal Ministry of Education and Research for financial support (grant #02S8345; given to F.E.-S.).
PY - 2008/11/1
Y1 - 2008/11/1
N2 - 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.
AB - 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.
KW - DNA double-strand break repair
KW - RAD6 group
KW - Ubiquitylation
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U2 - 10.1016/j.dnarep.2008.07.016
DO - 10.1016/j.dnarep.2008.07.016
M3 - Article
C2 - 18722556
AN - SCOPUS:53149134255
SN - 1568-7864
VL - 7
SP - 1893
EP - 1906
JO - DNA Repair
JF - DNA Repair
IS - 11
ER -