College of Life Sciences, Capital Normal University, Beijing 100048, China, Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon S7N 5E5, Canada and Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, Chiba 263-8555, Japan.
Nucleic Acids Res. 2013 Aug;41(15):7356-69. doi: 10.1093/nar/gkt542. Epub 2013 Jun 12.
In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.
针对复制阻断损伤，增殖细胞核抗原（PCNA）可在 K164 残基处被顺序泛素化，从而导致两种 DNA 损伤耐受模式，即跨损伤 DNA 合成（TLS）和无差错损伤旁路。尽管大多数报道的数据支持这样一种模型，即单泛素化 PCNA 增强其与 TLS 聚合酶的亲和力，从而将它们招募到损伤部位，但这一模型也受到了一些观察结果的挑战。在这项研究中，我们以可诱导的方式在培养的人类细胞中表达 PCNA-164R 和泛素（UB）融合基因，试图模拟 PCNA 的单泛素化。结果发现，N 端和 C 端 PCNA•Ub 融合体的表达均赋予细胞对紫外线（UV）诱导的 DNA 损伤显著的耐受能力。令人惊讶的是，Polη（一种专门用于绕过 UV 诱导的嘧啶二聚体的 TLS 聚合酶）的消耗并没有改变 PCNA•Ub 赋予的耐受能力。相反，另一种 TLS 聚合酶 Rev1（作为 TLS 复合物组装的支架）的消耗完全消除了 PCNA•Ub 介导的损伤耐受。当 RAD18 缺失消除内源性 PCNA 的 UV 诱导单泛素化时，类似的遗传相互作用也得到了证实。因此，PCNA•Ub 融合体绕过了 PCNA 单泛素化的要求，这些融合体赋予的 UV 损伤耐受依赖于 Rev1，但独立于 Polη。
