Formation of Neocentromere at a DSB Site

 描述(申请人提供)：组蛋白H3变体，着丝粒蛋白A(CENP-A)，也定位于DNA的双链断裂(DSB)，意味着参与了对DNA损伤的反应。CENP-A通常是着丝粒特异染色质的组成部分，它构成了着丝粒组装的基础，着丝粒是一种蛋白质结构，包含许多保守的蛋白质复合体，充当染色体和纺锤体微管之间的界面。CENP-A在着丝粒识别和着丝粒组装中起着至关重要的作用，对着丝粒和着丝粒的功能是必不可少的，但为什么它应该被招募到DSB焦点尚不清楚。我们的工作假设是CENP-A被招募到DSB中，以创建一种DNA损伤特异形式的染色质，DNA损伤检查点/修复蛋白被招募到该染色质中。基于这一思想，我们提出了一个新的模型来描述未修复DSB的一个重要生存机制。在这个模型中，未修复的DNA损伤会产生可能包含重要遗传信息的无着丝粒染色体片段。为了稳定地遗传这些无着丝粒片段，必须在断裂的染色体片段上由CENP-A蛋白标记的DSB处形成新的着丝粒。在有丝分裂过程中，新着丝粒促进微管附着和适当的分离。我们假设CENP-A在DSB位点的掺入可以导致新着丝粒的形成，以及在化疗或放射治疗导致DSB修复失败后细胞的存活。 这项提案的具体目标是确定中欧国家方案--A在复员方案中的作用。目的1.确定CENP-A在DSB位点的定位意义。我们将DSB引入CENP-A缺失的细胞的染色体中，然后分析DSB位点的DNA损伤检查点活性和DNA修复动力学，并确定是否取消了已知的DNA损伤检查点/修复蛋白到DSB位点的招募。此外，我们将测量DNA修复活性，以及CENP-A缺失细胞的存活率。目的2.确定DSB部位是否有新着丝粒形成。我们假设一个新着丝点或一个“假”新着丝点以CENP-A依赖的方式在DSB位点组装。在DSB上已经发现了一些纺锤体组装的检查点组件，但尚不清楚是否组装了一个能够捕获微管的功能性动粒。为了验证这一假设，我们将进行免疫荧光显微镜检测所有动粒成分。我们还将研究当DDR通路失败时，新着丝粒的形成是否增加。这些研究将为CENP-A在细胞存活中的作用提供新的见解。 该研究项目将确立CENP-A在DDR中的新角色，并重塑DNA损伤信号通路的图谱。如果新着丝粒的形成发生在DSB位点，它将代表一种以前未知的机制，以保护细胞免受DNA损伤导致的基因组不稳定的影响。

 DESCRIPTION (provided by applicant): The histone H3 variant, centromere protein A (CENP-A), also localizes to DNA at double-strand breaks (DSBs), implying involvement in responses to DNA damage. CENP-A is normally a constituent of centromere specific chromatin, which forms the foundation for assembly of the kinetochore, a proteinaceous structure comprising a number of conserved protein complexes, that serve as the interface between chromosomes and spindle microtubules. CENP-A plays a crucial role in centromere identity and kinetochore assembly, and is essential to centromere and kinetochore functions, but why it should be recruited to DSB foci is not known. Our working hypothesis is that CENP-A is recruited to DSBs to create a DNA damage-specific form of chromatin to which DNA-damage checkpoint/repair proteins are recruited. Based on this idea, we propose a new model to describe an important mechanism of survival of unrepaired DSBs. In this model, unrepaired DNA damage produces acentric chromosome fragments that may harbor essential genetic information. To stably inherit these acentric fragments, a neocentromere must be formed at the DSB site marked by CENP-A protein on the broken chromosome fragment. The neocentromere facilitates microtubule attachment and proper segregation during mitosis. We hypothesize that incorporation of CENP-A at a DSB site can result in the formation of a neocentromere, and survival of cells following the failure to repair DSBs induced by chemotherapeutic agents or radiation therapy. The specific objective of this proposal is to determine the role of CENP-A in the DDR. Aim 1. Determine significance of CENP-A localization at DSB sites. We will introduce DSBs into chromosomes in cells depleted of CENP-A, then analyze DNA damage checkpoint activity and DNA repair kinetics at the DSB site, and determine if recruitment of known DNA damage checkpoint/repair proteins to DSB sites is abrogated. Furthermore, we will measure DNA repair activity, and survival in cells depleted of CENP-A. Aim 2. Determine if neocentromere formation occurs at the site of DSBs. We hypothesize that a neocentromere or a "pseudo"-neocentromere assembles at the DSB site in a CENP-A-dependent manner. Some spindle assembly checkpoint components have been identified at DSBs, but it is unknown whether a functional kinetochore, which can capture microtubules, is assembled. To test this hypothesis, we will perform immunofluorescence microscopy to detect all the kinetochore components. We will also examine whether neocentromere formation is increased when DDR pathways fail. These studies will provide novel insight into the role of CENP-A in cell survival. The research project will establish a novel role of CENP-A in the DDR, and reshape the map of the DNA-damage signaling pathway. If neocentromere formation occurs at the DSB site, it will represent a previously unknown mechanism for protecting cells from genome instability in response to DNA damage.