Purification of proteins on newly synthesized DNA using iPOND.
Purification of proteins on newly synthesized DNA using iPOND.
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DOI：
10.1007/978-1-4939-1680-1_10
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Cortez, David
中科院分区：
文献类型：
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作者：
Dungrawala, Huzefa;Cortez, David
复制体是一种大型蛋白质机器，包含完成DNA复制所需的多种酶活性。除了复制DNA所需的解旋酶和聚合酶外，复制体还包含诸如DNA甲基转移酶、组蛋白伴侣和染色质修饰酶等蛋白质，以便将DNA复制与染色质沉积和表观遗传密码的建立联系起来。此外，由于模板DNA链常常包含DNA损伤或其他对复制机制的阻碍，复制应激反应蛋白会与复制体结合，以稳定、修复和重新启动停滞的复制叉。完成这些任务需要数百种蛋白质。识别这些蛋白质、监测它们的翻译后修饰以及了解它们的活性是如何协调的，对于理解基因组和表观基因组在每个细胞分裂周期中如何快速、完全和准确地复制是至关重要的。在此我们描述一种更新的iPOND（新生DNA上蛋白质的分离）方法来促进这些分析。
The replisome is a large protein machine containing multiple enzymatic activities needed to complete DNA replication. In addition to helicase and polymerases needed for copying the DNA, the replisome also contains proteins like DNA methyltransferases, histone chaperone and chromatin modifying enzymes to couple DNA replication with chromatin deposition and establishment of the epigenetic code. In addition, since template DNA strands often contain DNA damage or other roadblocks to the replication machinery, replication stress response proteins associate with the replisome to stabilize, repair and restart stalled replication forks. Hundreds of proteins are needed to accomplish these tasks. Identifying these proteins, monitoring their post-translational modifications, and understanding how their activities are coordinated is essential to understand how the genome and epigenome are duplicated rapidly, completely, and accurately every cell division cycle. Here we describe an updated iPOND (isolation of proteins on nascent DNA) method to facilitate these analyses.