Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells.
After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.
Atsuko
A
Niimi
Stephanie
S
Brown
Simone
S
Sabbioneda
Patricia L
PL
Kannouche
Andrew
A
Scott
Akira
A
Yasui
Catherine M
CM
Green
Alan R
AR
Lehmann
D002453:Cell Cycle; D002460:Cell Line; D006801:Humans; D008741:Methyl Methanesulfonate; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D025801:Ubiquitin / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; D008741:Methyl Methanesulfonate