Human DNA Replication Machines: Structure-Function of Polymerase Alpha-Primase

人类 DNA 复制机器：聚合酶 α-引物酶的结构-功能

• 批准号: 9548000
• 负责人: Tahir H Tahirov
• 金额: $ 13.1万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9548000
• 关键词: Active Sites; Adult; Affect; Amino Acid Substitution; Amino Acids; Area; Binding; Binding Sites; Biochemical; Biochemical Reaction; Biological; Biological Assay; Biology; Biophysics; C-terminal; Catalytic Domain; Cells; Complex; Crystallization; DNA; DNA Binding; DNA Polymerase I; DNA Primase; DNA Primers; DNA biosynthesis; DNA polymerase alpha-primase; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Disease; Drug Design; Drug Targeting; Eukaryota; Family; Genes; Genome; Genome Stability; Genomic Instability; Goals; Hand; Health; Human; Knowledge; Length; Life; Link; Maps; Medicine; Methods; Monitor; Movement; Mutation; Polymerase; Polymers; Positioning Attribute; Primer Extension; Process; Property; Proteins; RNA; RNA chemical synthesis; RNA primers; Reaction; Regulation; Ribonucleotides; Roentgen Rays; Role; Site; Structure; Surface; Surface Plasmon Resonance; Testing; Transfer RNA; Viral; X-Ray Crystallography; Yeast Model System; Yeasts; comparative; design; experimental study; human DNA; in vivo; insight; mutant; public health relevance; single molecule; tumor progression; virtual; yeast two hybrid system

DESCRIPTION (provided by applicant): The initiation of DNA synthesis and its regulation is a fundamental process of biology that impacts virtually every aspect of human health. Proper replication determines the fate of cells during early development and throughout adult life. DNA polymerases cannot synthesize DNA without a primer, and primase is the specialized RNA polymerase capable of de novo synthesis of short RNA primers during replication. In eukaryotes, primase functions within a heterotetrameric primase-DNA polymerase alpha (pol alpha) complex. This complex is uniquely capable of switching from the synthesis of RNA by primase to the synthesis of DNA by pol alpha. The synthesized RNA-DNA primer is required for further DNA synthesis by the major replicative DNA polymerases. In humans, the primase component consists of a small catalytic subunit (p49) and a large subunit (p58), and pol alpha is comprised of a catalytic subunit (p180) and an accessory subunit B (p70). The concerted actions of primase and pol alpha are critical for accurate genome duplication. Malfunction of primase-pol alpha complex causes global genome instability and is linked to the onset and progression of cancer and other diseases. Currently, the details for primase-pol alpha complex organization and function, including the mechanisms of unit size RNA primer synthesis and subsequent internal transfer to pol alpha are very limited. The goal of our project is to determine the structural basis of human primase-pol alpha complex function and reveal the biological consequences of alterations in this complex. To achieve our goal we will determine the mechanism of unit-length RNA primer synthesis and counting by human primase (Aim 1), the structural and functional consequences of primase integration into the pol alpha complex (Aim 2), and the mechanism of substrate switch from primase to pol alpha (Aim 3). Our studies will involve a variety of methods: X-ray crystallography, small angle X-ray scattering (SAXS), surface plasmon resonance (SPR), single molecule experiments, yeast two-hybrid and polymerase reactions assays. We also will examine the in vivo impact of mutations affecting primase-pol alpha activities on genome stability in a yeast model system.

描述（由申请人提供）：DNA 合成的启动及其调节是生物学的基本过程，几乎影响人类健康的各个方面。正确的复制决定了细胞在早期发育和整个成年生活中的命运。 DNA聚合酶在没有引物的情况下无法合成DNA，引物酶是一种特殊的RNA聚合酶，能够在复制过程中从头合成短RNA引物。在真核生物中，引物酶在异四聚体引物酶-DNA 聚合酶 α (pol α) 复合物中发挥作用。该复合物具有独特的能力，能够从引物酶合成 RNA 转换为 pol α 合成 DNA。合成的 RNA-DNA 引物是主要复制 DNA 聚合酶进一步合成 DNA 所必需的。在人类中，引物酶成分由小催化亚基 (p49) 和大亚基 (p58) 组成，pol α 由催化亚基 (p180) 和辅助亚基 B (p70) 组成。引物酶和 pol α 的协同作用对于基因组的精确复制至关重要。引物酶-pol α 复合物的故障会导致全球基因组不稳定，并与癌症和其他疾病的发生和进展有关。目前，引物酶-pol α复合物组织和功能的细节，包括单位大小RNA引物合成和随后内部转移到pol α的机制非常有限。我们项目的目标是确定 人引物酶-pol α 复合物功能的结构基础，并揭示该复合物改变的生物学后果。为了实现我们的目标，我们将确定单位长度 RNA 引物合成和人引物酶计数的机制（目标 1）、引物酶整合到 pol α 复合物中的结构和功能后果（目标 2）以及底物从引物酶转换为 pol α 的机制（目标 3）。我们的研究将涉及多种方法：X射线晶体学、小角X射线散射（SAXS）、表面等离子共振（SPR）、单分子实验、酵母双杂交和聚合酶反应测定。我们还将研究影响 primase-pol α 活性的突变对酵母模型系统中基因组稳定性的体内影响。

项目成果

Two steps forward, one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers.

• DOI: 10.1016/j.dnarep.2014.01.013
• 发表时间: 2014-08
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Spies, Maria

Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy.

• DOI: 10.1021/nl502890g
• 发表时间: 2014-10-08
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Ghoneim M;Spies M
• 通讯作者: Spies M