Rodrigues Joana, Banks Peter, Lydall David
Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Newcastle upon Tyne, NE2 4HH, United Kingdom.
High Throughput Screening Facility, Newcastle Biomedicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, United Kingdom.
G3 (Bethesda). 2018 May 4;8(5):1807-1816. doi: 10.1534/g3.118.200172.
In mammalian cell culture, the Golgi apparatus fragment upon DNA damage. GOLPH3, a Golgi component, is a phosphorylation target of DNA-PK after DNA damage and contributes to Golgi fragmentation. The function of the yeast () ortholog of GOLPH3, Vps74, in the DNA damage response has been little studied, although genome-wide screens suggested a role at telomeres. In this study we investigated the role of Vps74 at telomeres and in the DNA damage response. We show that Vps74 decreases the fitness of telomere defective cells and contributes to the fitness of cells. Importantly, loss of Vps74 in cells exacerbates the temperature dependent growth defects of these cells in a Chk1 and Mec1-dependent manner. Furthermore, Exo1 reduces the fitness of cells suggesting that ssDNA contributes to the fitness defects of cells. Systematic genetic interaction analysis of , and cells suggests that causes a milder but similar defect to that seen in cells. cells have slightly shorter telomeres and loss of in or cells further shortens the telomeres of these cells. Interestingly, loss of Vps74 leads to increased levels of Stn1, a partner of Cdc13 in the CST telomere capping complex. Overexpression of Stn1 was previously shown to cause telomere shortening, suppression of and enhancement of growth defects, suggesting that increased levels of Stn1 may be the route by which Vps74 affects telomere function. These results establish Vps74 as a novel regulator of telomere biology.
在哺乳动物细胞培养中，高尔基体在DNA损伤时会发生片段化。高尔基体蛋白3（GOLPH3）是一种高尔基体成分，是DNA损伤后DNA依赖性蛋白激酶（DNA-PK）的磷酸化靶点，并促使高尔基体片段化。尽管全基因组筛选表明高尔基体蛋白3的酵母直系同源物Vps74在端粒处发挥作用，但对其在DNA损伤反应中的功能研究甚少。在本研究中，我们调查了Vps74在端粒及DNA损伤反应中的作用。我们发现，Vps74降低了端粒缺陷型细胞的适应性，并有助于[具体细胞类型]细胞的适应性。重要的是，[具体细胞类型]细胞中Vps74的缺失会以依赖Chk1和Mec1的方式加剧这些细胞的温度依赖性生长缺陷。此外，Exo1降低了[具体细胞类型]细胞的适应性，这表明单链DNA（ssDNA）导致了[具体细胞类型]细胞的适应性缺陷。对[具体细胞类型1]、[具体细胞类型2]和[具体细胞类型3]细胞进行的系统遗传相互作用分析表明，[具体细胞类型1]导致的缺陷比[具体细胞类型2]细胞中的缺陷更轻微但相似。[具体细胞类型1]细胞的端粒略短，[具体细胞类型2]或[具体细胞类型3]细胞中[具体基因]的缺失会进一步缩短这些细胞的端粒。有趣的是，Vps74的缺失导致CST端粒封盖复合物中Cdc13的伙伴蛋白Stn1水平升高。先前的研究表明，Stn1的过表达会导致端粒缩短、[具体基因]的抑制以及[具体细胞类型]生长缺陷的增强，这表明Stn1水平的升高可能是Vps74影响端粒功能的途径。这些结果确立了Vps74作为端粒生物学的一种新型调节因子。
