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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase
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Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase

机译：从人类DNA引发酶的4Fe-4S簇结构域的结构和功能相互作用洞察真核DNA引发

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摘要

DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionary conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/ dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints from NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated ssDNA and regulation of primase activity.
机译：DNA复制需要通过称为primase的酶引发DNA模板。尽管可从古细菌和细菌中获得DNA primase结构，但由于缺乏大亚基（p58C）独特的，高度保守的C末端调节结构域的结构，对高级真核生物中DNA priming的机制仍知之甚少。 。在这里，我们介绍了通过X射线晶体学确定为1.7-A分辨率的该结构域的结构。 p58C结构揭示了一种进化保守的4Fe-4S簇的新颖排列，该簇深深地埋在蛋白核中，与任何已知的蛋白结构都不相似。通过荧光各向异性测量分析DNA与p58C的结合，显示出对ss / dsDNA连接底物的强烈偏好。该方法与定点诱变相结合，以确认DNA的结合发生在p58C的独特碱性表面上。鉴定了p58C与复制蛋白A的中间亚基（RPA32C）的C-末端结构域的特异性相互作用，并通过等温滴定热分析和NMR对其进行了表征。来自NMR实验的限制用于驱动两个域的计算对接，并生成p58C-RPA32C复合物的模型。在一起，我们的结果解释了人类DNA primase突变体中的功能缺陷，并提供了对RPA包被的ssDNA上的primosome负载和对primase活性进行调节的见解。

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    Sivaraja Vaithiyalingam; rnEric M. Warren; rnBrandt F. Eichman; rnWalter J. Chazin;

  • 作者单位

    Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-8725 Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725;

    rnCenter for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725 Departments of Biological Sciences, Vanderbilt University, Nashville, TN 37232-8725;

    rnDepartment of Biochemistry, Vanderbilt University, Nashville, TN 37232-8725 Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725 Departments of Biological Sciences, Vanderbilt University, Nashville, TN 37232-8725;

    rnDepartment of Biochemistry, Vanderbilt University, Nashville, TN 37232-8725 Center for Structural Biology, Vanderbilt University, Nashville, TN 37232-8725 Chemistry, Vanderbilt University, Nashville, TN 37232-8725;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    polymerase; replisome; replication initiation; primed DNA; iron-sulfur cluster;

    机译：聚合酶复制复制起始;引物DNA铁硫团簇;

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