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Broken chromosome segregation during mitosis: a Drosophila model

有丝分裂过程中染色体分离断裂：果蝇模型

基本信息

批准号：
10708770
负责人：
Donald T. Fox
金额：
$ 30.99万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-22 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708770
关键词：
Amaze Area Behavior Biochemical Biochemistry Biological Biological Models Biology Cell Cycle Cell Death Cell Nucleus Cell division Cells Centromere Childhood Chromosomal Breaks Chromosome Condensation Chromosome Segregation Chromosome Structures Chromosomes Complex DNA DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA Structure DNA damage checkpoint DNA-Directed DNA Polymerase Data Defect Dependence Detection Development Disease Disseminated Malignant Neoplasm Drosophila genus Elasticity Exhibits FANCD2 protein Face Fanconi&apos s Anemia Genetic Genetic Epistasis Genetic Screening Genome Genomic Instability Goals Health Human Image Immunity Interphase Interphase Cell Laboratories Left Link Malignant Neoplasms Mammalian Cell Mediating Mitosis Mitotic Mitotic Chromosome Modeling Molecular Monoubiquitination Nature Outcome Pathway interactions Positioning Attribute Process Property Proteins Proteomics Rectum Recurrence Regulation Role Signal Pathway Site Source Structure System Testing Tissues Wing Work condensin in vivo innovation insight interest micronucleus prevent recruit rectal repaired response scaffold segregation targeted cancer therapy tumor progression ubiquitin ligase

项目摘要

Chromosome double strand breaks (DSBs) that evade DNA damage checkpoints can persist into mitosis. These DSBs are in danger of forming highly detrimental structures connected to genome shattering and tumor progression, collectively referred to as micronuclei. To find mechanisms that prevent micronuclei, we discovered that Drosophila papillar cells naturally inactivate DNA damage checkpoints, and as a result frequently exhibit DNA fragments in mitosis. These fragments lack centromeres (acentric DNA), yet remarkably segregate during papillar mitosis. This process prevents micronuclei and tissue development defects. The distinctive dependence of papillar tissue development on acentric DNA segregation holds promise to reveal fundamental responses to DSBs that persist into mitosis. From our combination of in vivo genetic screens, live imaging, and complementary biochemistry approaches with collaborators, we are poised to make unique conceptual advances in this area. This proposal leverages our expertise, new findings, and a genetically amenable Drosophila model to uncover regulation of broken chromosome segregation. The significance of our proposed work is evident in the frequent contribution of micronuclei to genome instability and the evolutionary conservation of the molecules studied, including the Alternative End Joining (Alt-EJ) repair protein DNA Polymerase Theta, conserved monoubiquitination of the DNA repair scaffold FancD2, and the ubiquitin ligase CRL4CDT2. The innovation of our approach derives from our model system that is evolutionarily wired to solve the challenge of frequent persistent broken chromosomes, and the enhanced in vivo genetic screening capability of our system. These advantages led to the preliminary data presented in this proposal. In Aim1, we will define the pathway leading to poleward segregation of acentric DNA. In this Aim, we will identify Pol Theta domains that function in acentric DNA segregation, pinpoint the extent to which Alt-EJ occurs in papillar cells with DSBs, and assess the role of FancD2 in regulating Pol Theta after DSBs. In Aim2, we will define the signaling pathway that promotes the transition from lagging to segregating acentric DNA. In this Aim, we will determine if CRL4CDT2 functions together with Pol Theta/FancD2 to promote acentric DNA segregation, uncover whether critical regulation of CRL4CDT2 activity or in papillar cells with DSBs, and assess if inactivity of interphase checkpoints leads to a requirement for CRL4CDT2 in segregating acentric DNA in a non-papillar cell context (wing cells). In Aim 3, we will determine how regulation of mitotic chromosome condensation contributes to segregation of acentric DNA fragments. We will build on biochemical, genetic, and protein localization data connecting CRL4CDT2 to the mitotic complex Condensin I. We will assess the role of CRL4CDT2 in regulating Condensin I localization during acentric DNA segregation and determine the role of a conserved CRL4CDT2 recognition sequence in Condensin I subunits. Collectively, our approach will uncover fundamental regulation of broken mitotic DNA and can inform on disease-relevant biology.

逃避DNA损伤检查点的染色体双链断裂（DSB）可以持续到有丝分裂。这些DSB有形成与基因组破碎和肿瘤相关的高度有害结构的危险这一过程统称为微核。为了找到防止微核的机制，我们发现果蝇的乳头状细胞自然地破坏DNA损伤检查点，因此经常表现出有丝分裂中的DNA片段。这些片段缺乏着丝粒（无着丝粒DNA），但在生长过程中显著分离。乳头状有丝分裂。这一过程可防止微核和组织发育缺陷。独特的依赖性无着丝粒DNA分离的乳头状组织发育的承诺，揭示基本的反应，持续到有丝分裂的DSB。从我们结合体内基因筛选，活成像，和互补与合作者的生物化学方法，我们准备在这一领域取得独特的概念进展。这项提议利用了我们的专业知识、新发现和一个遗传上适合的果蝇模型，揭示染色体分离破坏的调节。我们建议的工作的重要性在以下方面显而易见：微核对基因组不稳定性和分子进化保守性的频繁贡献研究，包括交替末端连接（Alt-EJ）修复蛋白DNA聚合酶θ，保守的 DNA修复支架FancD 2的单泛素化和泛素连接酶CRL 4CDT 2。我们的创新这种方法源自我们的模型系统，该模型系统在进化上被连接以解决频繁的持久性挑战。断裂的染色体，以及我们系统的增强的体内遗传筛选能力。这些优势导致了本提案中提出的初步数据。在目标1中，我们将定义通向极向的路径无着丝粒DNA的分离。在这个目标中，我们将鉴定在无着丝粒DNA中起作用的Pol Theta结构域分离，确定Alt-EJ在具有DSB的乳头状细胞中发生的程度，并评估FancD 2的作用在DSB之后调节Pol Theta。在Aim 2中，我们将定义促进这种转变的信号通路。从滞后到分离无着丝粒DNA。在这个目标中，我们将确定CRL 4CDT 2是否与Pol Theta/FancD 2促进无着丝粒DNA分离，揭示CRL 4CDT 2活性的关键调节或在具有DSB的乳头状细胞中，并评估间期检查点的不活动是否导致需要CRL 4 CDT 2 在分离无着丝粒DNA在非乳头状细胞的情况下（翼细胞）。在目标3中，我们将确定如何监管有丝分裂染色体的凝聚有助于无着丝粒DNA片段的分离。我们将建立在将CRL 4 CDT 2连接到有丝分裂复合体Condensin I的生化、遗传和蛋白定位数据。我们将评估CRL 4CDT 2在无着丝粒DNA分离过程中调节凝聚蛋白I定位的作用，确定一个保守的CRL 4CDT 2识别序列在凝聚蛋白I亚基中的作用。总体而言，我们这种方法将揭示有丝分裂DNA断裂的基本调控，并可以为疾病相关生物学提供信息。