Brun Jan, Chiu Roland K, Wouters Bradly G, Gray Douglas A
Ottawa Health Research Institute, Ottawa, ON K1H 8L6, Canada.
BMC Res Notes. 2010 Mar 30;3:85. doi: 10.1186/1756-0500-3-85.
The ubiquitin-based molecular switch dictating error free versus error prone repair has been conserved throughout eukaryotic evolution. A central component of this switch is the homotrimeric clamp PCNA, which is ubiquitinated in response to genotoxic stress allowing recovery of replication forks blocked at sites of DNA damage. The particulars of PCNA ubiquitination have been elucidated in yeast and to a further extent recently in human cells. However, gaps in the detailed mechanism and regulation of PCNA polyubiquitination still persist in human cells.
We expand upon several studies and show that PCNA is polyubiquitnated in normal skin fibroblasts, and that this ubiquitination is dependant on RAD18. Furthermore we define the types of DNA damage that induce ubiquitination on PCNA. Cisplatin, methylmethane sulphonate and benzo(a)pyrene-diol-epoxide induce the polyubiquitination of PCNA to the same extent as UV while polyubiquitination is not detected after X-ray treatment. Moreover, we show that ubiquitination of PCNA is not regulated by cell cycle checkpoint kinases ATM-Chk2 or ATR-Chk1. Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.
Our results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination.
基于泛素的分子开关决定无差错修复与易出错修复，这在整个真核生物进化过程中一直保守。该开关的核心成分是同三聚体钳PCNA，它在受到基因毒性应激时会发生泛素化，从而使在DNA损伤位点受阻的复制叉得以恢复。PCNA泛素化的具体细节已在酵母中阐明，最近在人类细胞中也有了更深入的研究。然而，在人类细胞中，PCNA多聚泛素化的详细机制和调控方面仍存在空白。
我们在多项研究的基础上进一步表明，PCNA在正常皮肤成纤维细胞中会发生多聚泛素化，且这种泛素化依赖于RAD18。此外，我们确定了诱导PCNA泛素化的DNA损伤类型。顺铂、甲磺酸甲酯和苯并(a)芘 - 二醇环氧化物诱导PCNA多聚泛素化的程度与紫外线相同，而在X射线处理后未检测到多聚泛素化。此外，我们表明PCNA的泛素化不受细胞周期检查点激酶ATM - Chk2或ATR - Chk1的调控。值得注意的是，我们报告PCNA多聚泛素化受USP1负调控。
我们的结果证明了PCNA多聚泛素化在人类细胞中的重要性，并确定了这种泛素化的关键调节因子。
