Broken chromosome segregation during mitosis: a Drosophila model

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。 这些双链断裂有形成与基因组破碎和肿瘤有关的高度有害结构的危险。 进展，统称为微核。为了找到防止微核的机制，我们发现 果蝇乳头细胞自然使DNA损伤检查点失活，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 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 关于无着丝粒DNA分离的乳头组织发育的研究有望揭示 持续到有丝分裂的DSB。来自我们的体内基因筛查、实时成像和互补 随着生物化学方法与合作者的合作，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、新发现和遗传上可服从的果蝇模型来 揭示染色体分离断裂的规律。我们建议的工作的重要性显而易见地体现在 微核对基因组不稳定性和分子进化保守性的频繁贡献 已研究，包括Alt-EJ修复蛋白DNA聚合酶Theta，保守 DNA修复支架FANCD2和泛素连接酶CRL4CDT2的单素化。我们的创新之处 方法来自我们的模型系统，该模型系统进化地连接到解决频繁持久化的挑战 染色体断裂，以及我们系统增强的体内遗传筛选能力。这些优势 导致了这项提案中提出的初步数据。在Aim1中，我们将定义通向Poleward的路径 无着丝粒DNA的分离。在这个目标中，我们将确定在无着丝粒DNA中起作用的Pol Theta结构域 分离，确定Alt-EJ在DSB的乳头细胞中发生的程度，并评估FANCD2的作用 在调节DSB后的Pol Theta。在AIM2中，我们将定义促进转换的信号通路 从滞后到分离无着丝粒DNA。为此，我们将确定CRL4CDT2是否与POL一起发挥作用 Theta/FANCD2促进无着丝粒DNA分离，揭示CRL4CDT2活性的关键调控或 在患有DSB的乳头细胞中，评估间期检查点的不活跃是否导致对CRL4CDT2的需求 在非乳头细胞(翼细胞)中分离无着丝粒DNA。在目标3中，我们将确定监管如何 有丝分裂染色体的凝聚有助于无着丝粒DNA片段的分离。我们将在此基础上 将CRL4CDT2与有丝分裂复合体凝集素I联系起来的生化、遗传和蛋白质定位数据 将评估CRL4CDT2在调节无着丝粒DNA分离和凝聚素I定位中的作用 确定保守的CRL4CDT2识别序列在凝集素I亚基中的作用。总的来说，我们的 该方法将揭示断裂的有丝分裂DNA的基本规则，并可以为与疾病相关的生物学提供信息。

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.