Simpson Laura J, Sale Julian E
MRC Laboratory of Molecular Biology, Division of Protein & Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 2QH, UK.
DNA Repair (Amst). 2005 Apr 4;4(4):503-10. doi: 10.1016/j.dnarep.2004.12.002. Epub 2005 Jan 21.
The RAD6/RAD18 heterodimer promotes translesion synthesis via the monoubiquitination of the DNA sliding clamp, PCNA. In S. cerevisiae, a second complex, UBC13/MMS2/RAD5, can extend this single ubiquitin with a non-canonical lysine 63-linked chain. This polyubiquitination step is required for an error-free mode of bypass, possibly template switching by the stalled replication complex. Evidence of a role for the human homologue of MMS2, UBE2V2, in such a process has been inferred from the abrogation of ultraviolet light-induced gene conversion following antisense knockdown of the transcript in human fibroblasts. To ask whether a similar mechanism contributes to abasic site-induced immunoglobulin gene conversion, and to ascertain the role of UBE2V2 in the vertebrate DNA damage response we created a ube2v2 mutant of the chicken cell line DT40. Unlike budding yeast mms2, ube2v2 DT40 does not exhibit significant hypersensitivity to DNA damage, nor the elevated sister chromatid exchange seen in vertebrate rad18 mutants suggesting that UBE2V2 plays a minor or redundant role in RAD18 dependent DNA damage tolerance. In addition, both ube2v2 and rad18 DT40 display more or less normal levels of immunoglobulin gene conversion and, despite the important role played by RAD18 in DNA damage induced translesion synthesis, rad18 DT40, unlike rev1 DT40, does not show a defect in non-templated immunoglobulin gene mutation. Together these data suggest that signalling through PCNA ubiquitination is not required for immunoglobulin diversification in DT40.
RAD6/RAD18异二聚体通过DNA滑动夹PCNA的单泛素化促进跨损伤合成。在酿酒酵母中，第二个复合物UBC13/MMS2/RAD5可以用非经典的赖氨酸63连接链扩展这个单泛素。这个多泛素化步骤是无错旁路模式所必需的，可能是停滞的复制复合物进行模板切换。从人成纤维细胞中转录本的反义敲低后紫外线诱导的基因转换被废除这一现象可以推断出人类MMS2同源物UBE2V2在这样一个过程中的作用证据。为了探究类似的机制是否有助于无碱基位点诱导的免疫球蛋白基因转换，并确定UBE2V2在脊椎动物DNA损伤反应中的作用，我们构建了鸡细胞系DT40的ube2v2突变体。与芽殖酵母mms2不同，ube2v2 DT40对DNA损伤没有表现出明显的超敏反应，也没有在脊椎动物rad18突变体中看到的姐妹染色单体交换增加，这表明UBE2V2在依赖RAD18的DNA损伤耐受中起次要或冗余作用。此外，ube2v2和rad18 DT40都显示出或多或少正常水平的免疫球蛋白基因转换，并且，尽管RAD18在DNA损伤诱导的跨损伤合成中起重要作用，但rad18 DT40与rev1 DT40不同，在非模板化免疫球蛋白基因突变方面没有缺陷。这些数据共同表明，在DT40中免疫球蛋白多样化不需要通过PCNA泛素化进行信号传导。
