Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803, USA.
Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Biochem Biophys Res Commun. 2013 Oct 4;439(4):443-8. doi: 10.1016/j.bbrc.2013.08.089. Epub 2013 Sep 5.
The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p-Rnr4p subunits and inactivation of negative regulators Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p-Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p-Rnr4p.
进化保守的 MEC1 检查点途径介导细胞周期停滞，并诱导包括 RNR（核糖核苷酸还原酶）基因和 HUG1（羟脲、紫外线和γ辐射）在内的基因的表达，以响应 DNA 损伤和复制停滞。Rnr 复合物的活性部分受到细胞质中 Rnr2p-Rnr4p 亚基的定位以及 DNA 损伤和羟基脲（HU）处理后负调节剂 Sml1p 和 Dif1p 的失活的控制。我们之前表明，HUG1 的缺失挽救了 mec1Δ的致死性，并抑制了 dun1Δ菌株。在这项研究中，多种方法证明了 Hug1p 对 DNA 损伤和 HU 处理的调节反应，并支持其作为 MEC1 途径的负效应物的作用。与我们的假设一致，当 HUG1 过表达时，野生型细胞对 DNA 损伤和 HU 敏感。Hug1 多克隆抗血清表明 HUG1 在芽殖酵母中编码一种蛋白质，并且其 MEC1 依赖性表达与 HU 处理后 Rnr3p 的快速诱导相比延迟。细胞生物学和亚细胞分级分离实验表明，Hug1p-GFP 在 HU 处理后定位于细胞质。Hug1p-GFP 的细胞质定位依赖于 MEC1 途径基因，并且与 Rnr2p-Rnr4p 的细胞质定位一致。综上所述，遗传相互作用、基因表达和定位研究支持了 Hug1p 通过与 Rnr2p-Rnr4p 的区室化作为 MEC1 检查点反应的负调节剂的新作用。
