Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
J Mol Biol. 2010 Feb 26;396(3):487-500. doi: 10.1016/j.jmb.2010.01.003. Epub 2010 Jan 11.
Translesion DNA synthesis is a mechanism of DNA damage tolerance, and mono-ubiquitination of proliferating cell nuclear antigen (PCNA) is considered to play a key role in regulating the switch from replicative to translesion DNA polymerases (pols). In this study, we analyzed effects of a replicative pol delta on PCNA mono-ubiquitination with the ubiquitin-conjugating enzyme and ligase UBE2A/HHR6A/RAD6A-RAD18. The results revealed that PCNA interacting with pol delta is a better target for ubiquitination, and PCNA mono-ubiquitination could be coupled with DNA replication. Consequently, we could reconstitute replication-coupled switching between pol delta and a translesion pol, pol eta, on an ultraviolet-light-irradiated template. With this system, we obtained direct evidence that polymerase switching reactions are stimulated by mono-ubiquitination of PCNA, depending on a function of the ubiquitin binding zinc finger domain of pol eta. This study provides a framework for detailed analyses of molecular mechanisms of human pol switching and regulation of translesion DNA synthesis.
跨损伤 DNA 合成是一种 DNA 损伤容忍机制，增殖细胞核抗原（PCNA）的单泛素化被认为在调节从复制到跨损伤 DNA 聚合酶（pols）的转换中发挥关键作用。在这项研究中，我们分析了复制 pol delta 对泛素连接酶和连接酶 UBE2A/HHR6A/RAD6A-RAD18 的 PCNA 单泛素化的影响。结果表明，与 pol delta 相互作用的 PCNA 是更好的泛素化靶标，并且 PCNA 单泛素化可以与 DNA 复制偶联。因此，我们可以在紫外线照射的模板上重建 pol delta 和跨损伤 pol pol eta 之间的复制偶联转换。通过该系统，我们获得了直接证据，表明聚合酶转换反应受 PCNA 的单泛素化刺激，这取决于 pol eta 的泛素结合锌指结构域的功能。本研究为详细分析人类 pol 转换的分子机制和跨损伤 DNA 合成的调控提供了框架。
