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

描述（由申请人提供）：DNA合成的起始及其调控是生物学的一个基本过程，几乎影响到人类健康的各个方面。适当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，而引物酶是一种特殊的RNA聚合酶，能够在复制过程中重新合成短RNA引物。在真核生物中，引物酶在异四聚体引物酶- dna聚合酶（pol α）复合体中起作用。这种复合物具有独特的功能，可以从引物酶合成RNA转变为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，引物酶成分由一个小的催化亚基（p49）和一个大的亚基（p58）组成，pol α由一个催化亚基（p180）和一个辅助亚基B （p70）组成。引物酶和pol - α的协同作用对精确的基因组复制至关重要。引物-pol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，关于引物-pol - α复合物的组织和功能，包括单位大小RNA引物合成和随后向pol - α内部转移的机制的细节非常有限。我们项目的目标是确定

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.