Dpb2 Integrates the Leading-Strand DNA Polymerase into the Eukaryotic Replisome
Dpb2 Integrates the Leading-Strand DNA Polymerase into the Eukaryotic Replisome
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DOI：
10.1016/j.cub.2013.02.011
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发表时间：
2013-04-08
期刊：
影响因子：
9.2
通讯作者：
Labib, Karim
中科院分区：
文献类型：
--
作者：
Sengupta, Sugopa;van Deursen, Frederick;Labib, Karim
背景：真核生物的复制体是基因组完整性的关键决定因素，其复杂的结构仍然没有得到很好的描述。一个中心悬而未决的问题是CDC45-MCM-gins解旋酶如何与DNA聚合酶epsilon相连，后者在复制分叉处合成前导链，也是一个重要的调控焦点。结果：在这里，我们使用发芽酵母来证明Pol epsilon的Dpb2亚单位(Dpb2NT)的一个保守的氨基末端结构域与Gins的Psf1成分相互作用，后者的独特的B结构域对于GINS复合体的组装是必不可少的，但对复制启动是必不可少的。我们发现，在CDC45-MCM-GINS解旋酶的组装过程中，Dpb2NT是必需的。此外，在内源Dpb2耗尽的情况下，过表达的Dpb2NT足以支持在起始过程中CDC45-MCM-gins解旋酶的组装。这就产生了一个缺乏DNA聚合酶epsilon的复制体，尽管细胞是有活力的，但它们的生长速度非常慢。最后，我们使用一种新的体外实验证明了Dpb2NT对于POL epsilon在启动后与复制体的相互作用是必不可少的。结论：这些发现表明Dpb2与Psf1的B结构域的关联在萌芽酵母的染色体复制过程中起着两个关键作用。首先，它是启动所必需的，因为它促进了GINS在新生分叉处被结合到CDC45-MCM-GINS解旋酶中。其次，它在启动后发挥着同样重要的作用，因为它将DNA聚合酶的前导链与复制体内的CDC45-MCM-GINS解旋酶联系起来。
Background: The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation.Results: Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol epsilon (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol epsilon to interact with the replisome after initiation.Conclusions: These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.