A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.
A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome.
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DOI：
10.1074/jbc.m112.368951
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发表时间：
2012-07-06
期刊：
影响因子：
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通讯作者：
Pellegrini L
中科院分区：
文献类型：
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作者：
Kilkenny ML;De Piccoli G;Perera RL;Labib K;Pellegrini L
背景：Primase与dna聚合酶α一起启动dna复制，构成真核复制体的一部分。结果：在Pollα中，Primase被一个具有重要功能的短基序所束缚。结论：将启动子酶与真核细胞复制体连接是保证DNA正常复制的关键。意义：Primase-Pollα相互作用的小分子抑制剂可能在抗肿瘤治疗中有价值。DNA聚合酶α-Primase复合体是真核复制体的重要组成部分。底物酶和PolDNA的催化亚基合成复合α-DNA引物，在复制分叉处启动领先和落后的dna链。通过POLα将启动子酶连接到真核复制体的物理基础和生理意义尚不清楚。我们在Polα的末端发现了一个保守的短基序，该基序对于酵母同源蛋白Pol1与底物酶的相互作用是至关重要的。我们发现Pol1的C-末端1452-1468残基的截断取消了与启动酶的相互作用，不变氨基酸Phe1463的丙氨酸突变也是如此。相反，跨越最后16个残基的Pol1肽以高亲和力结合Primase，而来自人Pol1α的等效肽以类似的方式结合Primase。这些体外数据在酵母细胞中的实验中得到了反映，因为在终止于1452位残基或具有F1463A突变的pol1细胞提取物中，Primiase不会与之相互作用。破坏启动子酶和Pollα之间的联系的能力使我们能够评估以这种方式将启动子酶拴在真核复制体上的生理意义。我们发现Pol1的F1463A突变使酵母细胞依赖于S阶段的检查点，而Pol1在1452位氨基酸的截断阻止了酵母细胞的增殖。这些发现表明，PolDNA将启动子酶连接到复制体对于真核细胞中α复制叉的正常作用是至关重要的。
Background: Primase initiates DNA replication together with DNA polymerase α and forms part of the eukaryotic replisome. Results: Primase is tethered by a short motif in pol α that is functionally important. Conclusion: Tethering primase to the eukaryotic replisome is critical for normal DNA replication. Significance: Small molecule inhibitors of the primase-pol α interaction might be valuable in antitumor therapies. The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452–1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe1463. Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.