Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada.
BMC Genet. 2012 May 7;13:36. doi: 10.1186/1471-2156-13-36.
The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK). In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS) leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress.
We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA) is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU) and to the base analogue 5-fluorouracil (5-FU) but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE) the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate.
Together these observations point to a role for DDK-mediated phosphorylation of Mcm2 in the response to replicative stress, including some forms of DNA damage. We suggest that phosphorylation of Mcm2 modulates Mcm2-7 activity resulting in the stabilization of replication forks in response to replicative stress.
真核细胞中的复制解旋酶由 minichromosome maintenance（Mcm）蛋白 2 至 7（Mcm2-7）组成，是细胞增殖调控的关键靶点。此外，它还受到复制应激的调节。靶向 Mcm2-7 的蛋白激酶之一是 Dbf4 依赖性激酶 Cdc7（DDK）。在之前的研究中，我们发现酿酒酵母 Mcm2 中的 DDK 磷酸化位点 S164 和 S170 的丙氨酸突变导致对咖啡因和甲基甲磺酸（MMS）敏感，这使我们认为 DDK 对 Mcm2 的磷酸化是应对复制应激所必需的。
我们在这里表明，缺乏 DDK 磷酸化位点的 mcm2 等位基因（mcm2AA）菌株也对核糖核苷酸还原酶抑制剂羟基脲（HU）和碱基类似物 5-氟尿嘧啶（5-FU）敏感，但对放射模拟药物博来霉素不敏感。我们筛选了出芽酵母非必需缺失集合，以寻找与 mcm2AA 的合成致死相互作用，并分离出包括参与基因组完整性和氧化应激控制的基因缺失。此外，与野生型相比，mcm2AA 菌株的自发突变率（由 CAN1 中的突变测量）增加，而在磷酸模拟等位基因（mcm2EE）中，突变率降低。这些结果表明，mcm2AA 菌株无法正确应对 DNA 损伤。我们通过筛选缺失集合以寻找 mcm2AA 对咖啡因敏感性的抑制子来研究这一点。分离出降低自发 DNA 损伤、增加同源重组或减缓复制叉的缺失。许多咖啡因敏感性的抑制子抑制了 mcm2AA 的其他表型，包括对遗传毒性药物的敏感性、RPA 焦点形成细胞的增加频率和突变率的增加。
这些观察结果共同表明，DDK 介导的 Mcm2 磷酸化在应对复制应激中发挥作用，包括某些形式的 DNA 损伤。我们认为，Mcm2 的磷酸化调节 Mcm2-7 的活性，导致复制叉在应对复制应激时稳定。
