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

基本信息

批准号：
8504521
负责人：
Tahir H Tahirov
金额：
$ 43.9万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8504521
关键词：
Active Sites Adult Affect Amino Acid Substitution Amino Acids Area Binding Binding Sites Biochemical Biochemical Reaction Biological Biological Assay Biology C-terminal Catalytic Domain Cells Complex 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 Targeting Eukaryota Family Genes Genome Genome Stability Genomic Instability Goals Hand Health Human Knowledge Lead Length Life Link Maps Medicine Methods Monitor Movement Mutation Polymerase Positioning Attribute Primer Extension Process Property Proteins RNA RNA chemical synthesis RNA primers Reaction Regulation Relative (related person)Ribonucleotides Roentgen Rays Role Site Solutions Structure Surface Surface Plasmon Resonance Testing Transfer RNA Viral X-Ray Crystallography Yeast Model System Yeasts base comparative design human DNA in vivo insight mutant public health relevance research study single molecule tumor progression yeast two hybrid system

项目摘要

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合成及其调节的启动是生物学的基本过程，实际上影响了人类健康的各个方面。正确的复制确定了早期发育和整个成人生活中细胞的命运。 DNA聚合酶在没有底漆的情况下无法合成DNA，而原始酶是能够在复制过程中从头合成短RNA引物的专门RNA聚合酶。在真核生物中，原始酶在异次二聚酶-DNA聚合酶α（pol alpha）复合物中的功能。该复合物具有独特的能力从原始酶从RNA的合成转变为Pol alpha的DNA的合成。通过主要的复制性DNA聚合酶进一步合成了合成的RNA-DNA引物。在人类中，原始酶成分由一个小的催化亚基（p49）和一个大亚基（p58）组成，而POL alpha由催化亚基（P180）和附件亚基B（p70）组成。原始酶和pol alpha的一致作用对于准确的基因组重复至关重要。培养基 - POLα络合物的故障会导致全球基因组不稳定性，并与癌症和其他疾病的发作和进展有关。当前，Primase-Pol Alpha复合组织和功能的细节，包括单位大小RNA引物合成的机制以及随后的内部转移到POL Alpha的机制非常有限。我们项目的目标是确定人类primase-polα复合功能的结构基础，并揭示了这种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引物合成和人类原始酶计数的机制（AIM 1），原始酶整合到Pol Alpha复合物中的结构和功能后果（AIM 2）以及底物开关的机制从底漆到pol alpha（AIM 3）。我们的研究将涉及多种方法：X射线晶体学，小角度X射线散射（SAXS），表面等离子体共振（SPR），单分子实验，酵母两杂化和聚合酶反应分析。我们还将检查影响酵母模型系统中影响原始酶-POLα活动对基因组稳定性的突变的体内影响。