Studies of DNA Licensing in Apicomplexa Parasites

现有的治疗方法，特别是针对疟疾的治疗方法，不断受到后天寄生虫药物的压力。 耐药性需要继续寻找新的治疗方法。尖复合体的特殊增殖周期 寄生虫与它们栖息的宿主有很大的不同，应该提供肥沃的土壤来供应活跃的 新目标层出不穷。为了实现这一承诺，我们需要更好地理解独特的结构和 寄生虫细胞分裂的分子特征。Apicomplexan的增殖已经适应了不同的宿主细胞 染色体复制周期，核复制的规模从几个到数百个不等 每天产生细胞核，这是无与伦比的细胞周期灵活性。如何通过变量保持保真度 染色体复制的轮回是Apicomplexa生物学的一大谜团，因为许多已知的 这些寄生虫缺乏多细胞真核生物中的DNA复制。此外，基本检查站 调节细胞周期转变的机制也鲜为人知。目前还不知道是什么控制着 G1期到S期承诺，S期进展，染色体分离还是什么对照允许的寄生虫 在分裂分裂过程中，在一种类型的染色体周期中放弃萌发，而在另一种类型的染色体周期中不放弃萌发 内生性多基因。在这个应用中，我们将研究几种携带致死点的弓形虫突变株 对正确的染色体复制和分离至关重要的蛋白质突变。当转换为高时 温度这些突变体在染色体复制中都遭受了非常相似的破坏。在目标1中，我们将 确定一种新的环蛋白(ECR1，染色体复制所必需的1)的分子功能 调节染色体复制和分离。我们将确定ECR1是否是发散的E3连接酶 并将这一机制在控制DNA复制方面的分子特征与已知的 染色体许可因子TOPO-II。我们还将研究ECR1在调节 速殖子细胞周期。ECR1与人细胞周期蛋白依赖性激酶2的弓形虫同源基因形成复合体 (TgCDK2)。ECR1/TgCDK2复合体出现在中心锥中，然后离开这个隔室 在S阶段成为独家核电。我们将描述这种相互作用的分子基础，并 确定ECR1功能是否需要这种伙伴关系。我们还将确定以下基本功能 TgCDK2的作用机制包括是否需要周期蛋白才能发挥作用，以及确定TgCDK2的蛋白底物 为了了解TgCDK2是如何调节速殖子S期进程的，我们对TgCDK2进行了研究。在……里面 目的2，我们将研究两个含有另外两个缺陷ECR因子(ECR2和3)的化学突变体 当突变寄生虫在高温下生长时，也会导致不受控制的DNA合成。ECR2和 ECR3是仅在Apicomplexa遗传谱系中保守的未知蛋白质。发现了 对染色体复制至关重要的顶端复合体特异因子支持我们的中心假设，即这些 古老的寄生虫已经进化出独特的分子机制来调节无性阶段的增殖。

Existing therapies, particularly against malaria, are under constant pressure from acquired parasite drug resistance requiring a continuing search for new treatments. The peculiar proliferative cycles of Apicomplexa parasites differ substantially from the hosts they inhabit and should offer fertile ground to supply an active pipeline of new targets. To fulfill this promise, we need a better understanding of the unique structural and molecular features of parasite cell division. Apicomplexan proliferation has adapted to different host cells using chromosome replication cycles that can vary in the scale of nuclear reduplication from a few to hundreds of nuclei produced per day, which is unparalleled cell cycle flexibility. How fidelity is preserved through variable rounds of chromosome replication is a major mystery of Apicomplexa biology as many known regulators of DNA replication in multicellular eukaryotes are missing in these parasites. Further, the basic checkpoint mechanisms that regulate the cell cycle transitions are also poorly understood. It is not known what controls G1 to S phase commitment, S phase progression, chromosome segregation or what controls allow the parasite to forgo budding in one type of chromosome cycle but not in another during the processes of schizogony and endopolygeny. In this application we will investigate several Toxoplasma mutants that carry lethal point mutations in proteins essential for proper chromosome replication and segregation. When shifted to high temperature these mutants all suffer very similar disruptions in chromosome replication. In Aim 1, we will define the molecular function of a novel RING protein (ECR1, essential for chromosome replication 1) in regulating chromosome replication and segregation. We will determine whether ECR1 is a divergent E3 ligase and compare the molecular features of this mechanism in controlling DNA replication to the known chromosome licensing factor Topo-II. We will also investigate an alternate role for ECR1 in regulating the tachyzoite cell cycle. ECR1 forms a complex with a Toxoplasma ortholog of human cyclin-dependent kinase 2 (TgCDK2). The ECR1/TgCDK2 complex appears in the centrocone and then leaves this compartment to become exclusively nuclear during S phase. We will characterize the molecular basis for this interaction and determine whether this partnership is required for ECR1 function. We will also determine the basic features of the TgCDK2 mechanism including whether it requires a cyclin for function and identify the protein substrates of TgCDK2 in order to understand how this kinase mechanism regulates the tachyzoite S phase progression. In Aim 2, we will investigate two chemical mutants harboring two other defective ECR factors (ECR2 and 3) that also cause uncontrolled DNA synthesis when mutant parasites are grown at high temperature. ECR2 and ECR3 are unknown proteins conserved only within Apicomplexa genetic lineages. The discovery of apicomplexan-specific factors essential for chromosome replication supports our central hypothesis that these ancient parasites have evolved unique molecular mechanisms to regulate asexual stage proliferation.