Cell Cycle Regulation by Checkpoints in Drosophila

果蝇检查点的细胞周期调节

• 批准号: 6740168
• 负责人: Tin Tin Su
• 金额: $ 25.01万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740168
• 关键词: DNA damage; DNA replication; Drosophilidae; apoptosis; arthropod genetics; cell cycle; cell growth regulation; chromatin; chromosome aberrations; cyclins; cytology; embryogenesis; ionizing radiation; mitotic spindle apparatus; mutant; salivary glands; tissue /cell culture; video microscopy

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.

描述（由申请人提供）：我们将确定检查点如何调节 细胞周期响应于受损或未完全复制的DNA的细胞周期 果蝇的发展。细胞提取物和单细胞系统的研究具有 确定了监测基因组DNA并调节细胞的检查点机制 循环进展，允许时间进行修复和复制。在一代中 生命周期，检查站必须在深刻发展的背景下运行 细胞周期调节的变化，例如废除差距阶段或 有丝分裂。我们正在使用果蝇Melanogaster来确定如何知道检查点 机制在体内生物发育过程中的功能，并发现 在其他系统中尚未看到的新型机制。我们的最初发现表明 这种不同的机制可以使有丝分裂的响应于受损的DNA陷入困境 果蝇胚胎发生的不同阶段。多个的差异使用 后生发展中的机制表明失去了调节机制 对于一种细胞类型而言，比另一种细胞类型更有害。这个简单的概念可能 帮助我们了解由人类疾病的组织特异性 丢失检查点或单元周期调节器，从而更好地设计 疗法。我们的计划包括4个目标。目标1）我们发现持续的阶段 通过在组织/细胞中电离辐射来抑制DNA损伤 幼虫中的特定方式。 01/s调节器中的突变体（例如RB和 E2F），G2/M调节剂（例如CDK1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质在抑制S相中的作用， 并确定为什么只有某些细胞显示此响应。目标2）我们有 发现辐照不仅抑制了进入有丝分裂的，而且还抑制了 通过果蝇胚胎的有丝分裂进展；预言变得延长，并 中期后期过渡延迟。分子的组合 遗传和细胞学方法将确定调节的机制 照射后通过有丝分裂进展。目标3）我们发现 不完整的DNA复制阻止了通过主轴从有丝分裂的出口 检查点蛋白质并最终导致凋亡。遗传的结合 和细胞学方法，以及染色体的实时成像和 有丝分裂纺锤体将用于发现连接不完整DNA的机制 复制到主轴检查点和细胞死亡。目标4）我们将筛选 通过现有的诱变果蝇突变体的能力 调节辐照后有丝分裂，S期和细胞死亡，以发现 适当反应DNA损伤所需的新基因。