Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5.

酿酒酵母（Saccharomyces cerevisiae）Fun30染色质重塑因子（Fun30 chromatin remodeler）近期被证实可促进DNA双链断裂（DNA double strand break, DSB）末端的长程切除，该过程依赖同源重组（homologous recombination, HR）。学界普遍认为，这是Fun30介导细胞抵抗DSB诱导剂喜树碱（camptothecin）的核心作用基础。本研究证实，Fun30同样可增强细胞对两类遗传毒物（genotoxins）的抗性，即甲基磺酸甲酯（methyl methanesulfonate, MMS）与羟基脲（hydroxyurea, HU），二者均可阻滞DNA复制进程。我们提供的证据表明，DNA末端切除（DNA end resection）参与了Fun30依赖的MMS抗性通路。另一方面，我们发现Fun30缺失可抑制缺失Rad5/Mms2/Ubc13依赖的无错DNA损伤耐受机制（Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism）的细胞对MMS与HU的敏感性。该抑制效应并非由DNA末端切除水平降低所致，且依赖核心HR组分Rad51。我们进一步证实，Fun30负向调控rad5Δ突变体细胞从MMS诱导的G2/M期阻滞中恢复的过程。综上，Fun30在DNA损伤修复中具备双重功能：其一为通过辅助DNA末端切除提升细胞对遗传毒性应激的抗性；其二为负向调控依赖Rad51但不依赖DNA末端切除的、对抗复制应激的通路，该功能在Rad5依赖的DNA损伤耐受功能受损时得以显现。此外，我们发现Fun30的推定泛素结合CUE结构域（ubiquitin-binding CUE domain）可限制其在Rad5缺失时阻碍细胞对MMS和HU耐受的能力。