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

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期的命运。DNA聚合酶在没有引物的情况下不能合成DNA，引物酶是能够在复制期间从头合成短RNA引物的专门RNA聚合酶。在真核生物中，引发酶在异源四聚体引发酶-DNA聚合酶α（pol α）复合物内起作用。这种复合物能够从引发酶合成RNA转变为聚合酶α合成DNA。合成的RNA-DNA引物是主要复制型DNA聚合酶进一步合成DNA所必需的。在人类中，引发酶组分由小催化亚基（p49）和大亚基（p58）组成，并且pol α由催化亚基（p180）和辅助亚基B（p70）组成。引发酶和pol alpha的协同作用对于准确的基因组复制至关重要。primase-pol alpha复合物的功能障碍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，primase-pol alpha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 alpha的机制非常有限。我们项目的目标是确定 人类primase-pol α复合体功能的结构基础，并揭示了该复合体改变的生物学后果。为了实现我们的目标，我们将确定单位长度的RNA引物合成和计数的机制，由人类引物酶（目标1），引物酶整合到聚合酶α复合物（目标2）的结构和功能的后果，和底物开关的机制从引物酶到聚合酶α（目标3）。我们的研究将涉及各种方法：X射线晶体学，小角X射线散射（SAXS），表面等离子体共振（SPR），单分子实验，酵母双杂交和聚合酶反应测定。我们还将研究在酵母模型系统中影响primase-pol α活性的突变对基因组稳定性的体内影响。

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.