Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo.
Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo.
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DOI：
10.1016/j.celrep.2019.12.101
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发表时间：
2020-02-04
期刊：
影响因子：
8.8
通讯作者：
Schildkraut CL
中科院分区：
文献类型：
--
作者：
Drosopoulos WC;Vierra DA;Kenworthy CA;Coleman RA;Schildkraut CL
人类DNA聚合酶δ (Pol δ)与DNA滑动钳增殖细胞核抗原（PCNA）形成全酶复合物，在基因组复制中发挥重要作用。在这里，我们利用活细胞单分子跟踪实时监测Pol δ全酶与基因组的相互作用。我们发现全酶在体内的组装和拆卸是高度动态和有序的。PCNA通常在Pol δ之前装载到基因组上。一旦组装，全酶在基因组上的寿命相对较短，这意味着合成冈崎片段可能需要多个Pol δ结合事件。在拆卸过程中，Pol δ解离通常先于PCNA卸载。我们还发现，全酶的催化亚基Pol δ p125维持在恒定的细胞水平，表明体内Pol δ水平的控制有活性机制。总之，我们的研究表明，Pol δ全酶在体内的组装和拆卸遵循一个主要的途径；然而，交替的路径被观察到。Drosopoulos等人报道了人类Pol δ全酶在体内基因组上的组装和拆卸是高度动态和有序的。他们发现Pol δ全酶复合物的组装和拆卸遵循体内的主要途径，也观察到替代途径。
Human DNA polymerase delta (Pol δ) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol δ holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered. PCNA generally loads onto the genome before Pol δ. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol δ binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol δ dissociation generally precedes PCNA unloading. We also find that Pol δ p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol δ levels in vivo. Collectively, our studies reveal that Pol δ holoenzyme assembly and disassembly follow a predominant pathway in vivo; however, alternate pathways are observed. Drosopoulos et al. report human Pol δ holoenzyme assembly and disassembly on the genome in vivo are highly dynamic and ordered. They find that assembly and disassembly of the Pol δ holoenzyme complex follow a predominant pathway in vivo, with alternate pathways also observed.
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