Cell Cycle Regulation by Checkpoints in Drosophila

描述(由申请人提供)：我们将决定检查站如何监管 细胞周期对受损或不完全复制的DNA的反应 果蝇的发育。对细胞提取物和单细胞系统的研究 确定了监控基因组DNA和调节细胞的检查点机制 循环进行，为修复和复制留出时间。在后生动物中 在生命周期中，检查站必须在深刻发展的背景下运作 细胞周期调控的变化，如间隙时相的取消或 有丝分裂。我们正在使用果蝇来确定已知的检查点 机制在生物体内发育过程中发挥作用，并揭示 新的机制在其他系统中还没有见过。我们的初步调查结果表明 不同的机制作用于延缓有丝分裂以响应受损的DNA 果蝇胚胎发育的不同阶段。多个词的区别用法 后生动物发育的机制表明，调节机制的丧失 对一种细胞类型的危害比对另一种细胞类型更大。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 失去一个检查点或细胞周期调节器，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阶段 通过组织/细胞中的电离辐射来响应DNA损伤而被抑制 在幼虫中以特定的周期方式。01/S调节剂中的突变体(如Rb和 E2F)、G2/M调节蛋白(如CDK1和有丝分裂周期蛋白)和DNA修复蛋白 将用于检测这些蛋白质在抑制S时相中的作用， 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，而且还抑制了 果蝇胚胎有丝分裂的研究进展；前期变长， 中后期过渡变迟缓。分子的结合， 遗传学和细胞学方法将确定调控机制 辐射后有丝分裂的研究进展。目标3)我们发现 不完全的DNA复制通过纺锤体阻止了有丝分裂的退出 检查点蛋白，并最终导致细胞凋亡。一种基因的组合 和细胞学方法，以及对染色体和 有丝分裂纺锤体，将用于揭示连接不完整DNA的机制 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来研究它们的能力 调节辐射后有丝分裂、S期和细胞死亡，以揭示 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.