Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.
Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.
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DOI：
10.1074/mcp.m115.048991
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发表时间：
2015-07
期刊：
影响因子：
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通讯作者：
Boisvert FM
中科院分区：
文献类型：
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作者：
Drissi R;Dubois ML;Douziech M;Boisvert FM
微小染色体维持复合物（MCM）蛋白是进行性DNA复制所必需的，并且是S期检查点的靶标。真核MCM复合物由六种蛋白质（MCM 2 -7）组成，它们形成具有DNA解旋酶活性的异六聚体环，装载在染色质上形成复制前复合物。一旦进入S期，解旋酶被激活并打开DNA双链体以在复制叉处募集DNA聚合酶。因此，MCM复合物在DNA复制过程中起着至关重要的作用，但最近的工作表明，MCM蛋白也可能参与DNA修复。在这里，我们采用了稳定同位素标记与氨基酸在细胞培养（SILAC）为基础的定量蛋白质组学与免疫沉淀的绿色荧光蛋白标记的融合蛋白，以确定蛋白质相互作用的MCM复合物，并量化的相互作用的变化，以响应DNA损伤。有趣的是，MCM复合物在与蛋白质如Importin 7、组蛋白伴侣ASF 1和DNA损伤后的染色体结构域解旋酶DNA结合蛋白3（CHD 3）的相互作用中显示出非常动态的变化。这些相互作用的变化伴随着MCM蛋白上特定位点的磷酸化和泛素化的增加以及MCM复合物与γ-H2 AX的共定位的增加，证实了这些蛋白质在DNA损伤位点的募集。总之，我们的数据表明，MCM蛋白参与染色质重塑响应DNA损伤。
The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.