Cell Cycle Regulation by Checkpoints in Drosophila

描述（由申请人提供）：我们将确定检查站如何监管 细胞周期对受损或不完全复制的DNA的反应， 果蝇发育。对细胞提取物和单细胞系统的研究 确定了监测基因组DNA和调节细胞的检查点机制， 周期进展，为修复和复制留出时间。在一个后生动物 生命周期，检查站必须在深刻的发展背景下运作， 细胞周期调节的变化，如间隙期的消除，或 分裂。我们用黑腹果蝇来确定 机制在体内生物发育过程中发挥作用，并揭示 在其他系统中尚未发现的新机制。我们的初步发现表明 不同的机制对有丝分裂的停止做出反应， 果蝇胚胎发育的不同阶段。multiple的区别使用 后生动物发育的机制表明， 对一种细胞类型比对另一种更有害。这个简单的概念可能 帮助我们了解人类疾病的组织特异性， 检查点或细胞周期调节因子的丢失，从而设计出更好的 治疗我们的计划包括四个目标。目的1）我们发现，正在进行的S期 响应于组织/细胞中电离辐射的DNA损伤而被抑制 周期特定的方式在幼虫。01/S调节子中的突变体（例如Rb和 E2 F）、G2/M调节因子（例如Cdk 1和有丝分裂细胞周期蛋白）和DNA修复蛋白 将用于测定这些蛋白在S期抑制中的作用， 并确定为什么只有某些细胞表现出这种反应。目标2）我们有 发现辐射不仅抑制了有丝分裂的进入， 在果蝇胚胎中通过有丝分裂的进展;前期变得延长， 中期和后期的转换变得延迟。一种分子， 遗传学和细胞学方法将确定调节 照射后通过有丝分裂进行。目标3）我们发现， 不完全的DNA复制通过纺锤体阻断了有丝分裂的出口 检查点蛋白，并最终导致细胞凋亡。基因的组合 和细胞学方法，以及染色体的实时成像， 有丝分裂纺锤体，将被用来揭示机制，连接不完整的DNA 复制到纺锤体检查点和细胞死亡。目标4）我们将筛选 通过现有的突变敏感的果蝇突变体， 调节辐射后的有丝分裂、S期和细胞死亡， 对DNA损伤做出适当反应所需的新基因。

DESCRIPTION (provided by applicant): We will determine how checkpoints regulate the cell cycle in response to damaged or incompletely replicated DNA during Drosophila development. Studies in cell extracts and unicellular systems have identified checkpoint mechanisms that monitor genomic DNA and modulate cell cycle progression, allowing time for repair and replication. During a metazoan life cycle, checkpoints must operate in the context of profound developmental changes in cell cycle regulation such as the abolishment of gap phases or mitosis. We are using Drosophila melanogaster to determine how known checkpoint mechanisms function during organismal developmental in vivo, and to uncover novel mechanisms not yet seen in other systems. Our initial findings indicate that different mechanisms act to stall mitosis in response to damaged DNA at different stages of Drosophila embryogenesis. The differential use of multiple mechanisms in metazoan development suggests that loss of a regulatory mechanism is more detrimental for one cell type than for another. This simple notion may help us to understand the tissue-specificity of human diseases that result from the loss of a checkpoint or a cell cycle regulator, and thus to design better therapies. Our plans include 4 aims. Aim 1) We have found that ongoing S phase is inhibited in response to DNA damage by ionizing radiation in a tissue/cell cycle specific manner in the larvae. Mutants in 01/S regulators (e.g Rb and E2F), G2/M regulators (e.g. Cdk1 and mitotic cyclins) and DNA repair proteins will be used to assay the role of these proteins in the inhibition of S phase, and to determine why only certain cells show this response. Aim 2) We have found that irradiation inhibited not only the entry into mitosis but also the progress through mitosis in Drosophila embryos; prophase becomes lengthened and metaphase anaphase transition becomes delayed. A combination of molecular, genetic and cytological approaches will identify mechanisms that regulate progress through mitosis after irradiation. Aim 3) We have found that incomplete DNA replication blocks the exit from mitosis via a spindle checkpoint protein and ultimately leads to apoptosis. A combination of genetic and cytological approaches, together with live imaging of chromosomes and mitotic spindles, will be used to uncover mechanisms that link incomplete DNA replication to the spindle checkpoint and to cell death. Aim 4) We will screen through existing mutagensensitive Drosophila mutants for their ability to regulate mitosis, S phase and cell death after irradiation, in order to uncover novel genes needed for proper response to DNA damage.