Niimi Atsuko, Brown Stephanie, Sabbioneda Simone, Kannouche Patricia L, Scott Andrew, Yasui Akira, Green Catherine M, Lehmann Alan R
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.
Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16125-30. doi: 10.1073/pnas.0802727105. Epub 2008 Oct 9.
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.
在暴露于阻碍复制叉进程的DNA损伤剂后，增殖细胞核抗原（PCNA）的单泛素化介导了从复制性DNA聚合酶到跨损伤合成DNA聚合酶的转换。我们发现，在人类细胞中，PCNA会响应甲磺酸甲酯、丝裂霉素C以及紫外线而发生单泛素化，尽管动力学有所不同，但对博来霉素或喜树碱无响应。环丁烷嘧啶二聚体是紫外线照射后大多数PCNA泛素化事件的原因。PCNA未能发生泛素化会导致对紫外线和甲磺酸甲酯的显著敏感性增加，但对喜树碱或博来霉素则不然。PCNA泛素化依赖于复制蛋白A（RPA），但独立于ATR介导的检查点激活。紫外线照射后，去泛素化酶USP1的消失与PCNA泛素化的存在之间存在时间相关性，但化学诱变剂处理后未发现这种相关性。通过使用表达光裂合酶的细胞，我们能够去除紫外线损伤，并且我们发现损伤去除后PCNA泛素化仍持续数小时。我们提出了一个复制叉后跨损伤合成的模型来解释泛素化PCNA的持续存在。
