Proteomics of mitotic sister chromatid junctions in response to DNA replication stress

有丝分裂姐妹染色单体连接响应 DNA 复制应激的蛋白质组学

• 批准号: 412943020
• 负责人: Dr. Markus Räschle
• 金额: --
• 依托单位: Fachgebiet Molekulare Biotechnologie und Systembiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/412943020?language=en
• 关键词: Proteomics; mitotic; sister; chromatid; junctions

Replication stress is a key driver of genomic instability and has been linked to cancer and other human diseases. Under mild replication stress, which is relevant in disease settings, cells can enter mitosis despite an incompletely replicated genome. At under-replicated sites, sister-chromatids remain linked through distinct mitotic structures called ultra-fine bridges (UFBs), which become visible as cells start to segregate their chromosomes. Several proteins localize to UFBs where they contribute to the resolution of the structures. One of these proteins is the BLM helicase, whose inactivation gives rise to Bloom syndrome, a disorder characterized by genome instability and cancer predisposition. Besides the putative role in UFB resolution, BLM also plays pivotal roles in other genome maintenance pathways, such as the resection of DNA ends, the resolution of DNA recombination intermediates or the reactivation of stalled replication forks. It therefore remains difficult to determine the precise contribution of UFB resolution pathways to the maintenance of genome integrity. We have addressed this question and generated chromosomally stable HCT116 cells, in which the BLM protein can be rapidly depleted. For our project, we propose to characterize how BLM depletion in different phases of the cell cycle affects genome stability. Using live and fixed cell microscopy, we will follow synchronized cells through S-phase and compare their fate in the presence or absence of the BLM helicase. We hypothesize that defects in UFB resolution will increase chromosome mis-segregation. To test this, we will evaluate the progression of mitosis and analyse karyotypic changes using NGS right after the first mitosis in the absence of BLM. To further characterize UFB resolution pathways, we will continue with our established chromatin immunoprecipitation mass spectrometry assays to identify novel proteins interacting with the BLM helicase or other known UFB associated proteins on mitotic chromosomes. These comprehensive analyses will provide new insights into the processes that mitigate problems resulting from replication stress and contribute to faithful chromosome segregation.

复制应激是基因组不稳定的关键驱动因素，并与癌症和其他人类疾病有关。在温和的复制压力下，尽管基因组复制不完全，但细胞仍可进入有丝分裂。在复制不足的部位，姐妹染色单体仍然通过不同的有丝分裂结构连接在一起，这种结构称为超细桥(UFBs)，当细胞开始分离它们的染色体时，这种结构变得可见。有几种蛋白质定位于UFBs，在那里它们有助于结构的解析。其中一种蛋白质是BLM解旋酶，它的失活会导致Bloom综合征，一种以基因组不稳定和癌症易感性为特征的疾病。除了在UFB的分解中发挥作用外，BLM还在其他基因组维持途径中发挥关键作用，如DNA末端的切除、DNA重组中间产物的分解或停滞的复制叉子的重新激活。因此，仍然很难确定UFB分解途径对维持基因组完整性的确切贡献。我们已经解决了这个问题，并产生了染色体稳定的HCT116细胞，在这种细胞中，BLM蛋白可以迅速耗尽。在我们的项目中，我们建议表征细胞周期不同阶段的BLM耗尽如何影响基因组稳定性。使用活细胞显微镜和固定细胞显微镜，我们将跟踪同步细胞通过S期，并比较它们在有或没有博莱姆解旋酶的情况下的命运。我们假设UFB分辨率的缺陷会增加染色体的错误分离。为了验证这一点，我们将评估有丝分裂的进展，并在没有BLM的情况下，在第一次有丝分裂后立即使用NGS分析核型变化。为了进一步确定UFB的分解途径，我们将继续使用我们建立的染色质免疫沉淀质谱分析来鉴定与BLM解旋酶相互作用的新蛋白或有丝分裂染色体上其他已知的UFB相关蛋白。这些全面的分析将为减轻复制压力引起的问题并有助于忠实的染色体分离的过程提供新的见解。