Department of Environment and Primary Prevention, Section of Molecular Epidemiology, Rome, Italy.
DNA Repair (Amst). 2012 Jun 1;11(6):579-86. doi: 10.1016/j.dnarep.2012.03.004. Epub 2012 Apr 7.
To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.
为了维持基因组完整性，细胞必须对各种外源和内源的 DNA 损伤源做出适当的反应。DNA 完整性是通过 DNA 损伤反应机制和 DNA 修复的协调作用来维持的。此外，还有损伤容忍机制，如跨损伤合成（TLS），这对于 DNA 损伤后存活是很重要的，但潜在地容易出错。在这里，我们通过使用瞬时短发夹 RNA 干扰来沉默人细胞中的 DNA 聚合酶 κ（pol κ），研究了 pol κ 在烷基化损伤的 TLS 中的作用。我们表明，人 pol κ 在不显著影响突变性的情况下，对甲基亚硝脲（MNU）相关的细胞毒性具有显著的保护作用。当 pol κ 下调时，MNU 诱导的细胞毒性增加受到 O6-甲基鸟嘌呤 DNA 甲基转移酶水平的影响，并且当错配修复（MMR）有缺陷时，完全被消除。在 MNU 处理后，细胞周期谱不受 pol κ 状态的影响。pol κ 的下调导致第二次有丝分裂的起始严重延迟，这完全依赖于 O6-甲基鸟嘌呤（O6-meGua）损伤的存在。在 MNU 暴露后，在没有 pol κ 的情况下，姐妹染色单体交换的频率不受影响，而 RAD 51 焦点的诱导增加。我们提出，pol κ 通过结合 RAD 51 来恢复含有由 O6-meGua 产生的单链缺口的复制双链的完整性，从而部分保护人细胞免受 MMR 依赖的 O6-meGua 损伤的细胞毒性。
