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

复制压力是基因组不稳定性的关键驱动因素，并与癌症和其他人类疾病有关。在与疾病环境相关的轻度复制应激下，尽管基因组复制不完全，细胞仍可以进入有丝分裂。在复制不足的位点，姐妹染色单体通过称为超细桥（UFB）的独特有丝分裂结构保持连接，当细胞开始分离染色体时，这种结构变得可见。几种蛋白质定位于UFB，在那里它们有助于结构的解析。这些蛋白质之一是BLM解旋酶，其失活引起布卢姆综合征，这是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的解析中的假定作用之外，BLM还在其他基因组维持途径中发挥关键作用，例如DNA末端的切除、DNA重组中间体的解析或停滞的复制叉的再激活。因此，仍然难以确定UFB解析途径对维持基因组完整性的精确贡献。我们已经解决了这个问题，并产生了染色体稳定的HCT 116细胞，其中BLM蛋白可以迅速耗尽。对于我们的项目，我们建议表征BLM在细胞周期的不同阶段的消耗如何影响基因组稳定性。使用活细胞和固定细胞显微镜，我们将通过S期跟踪同步化细胞，并比较它们在BLM解旋酶存在或不存在下的命运。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。为了进一步表征UFB解析途径，我们将继续使用我们已建立的染色质免疫沉淀质谱分析，以鉴定与BLM解旋酶或有丝分裂染色体上其他已知UFB相关蛋白相互作用的新蛋白。这些全面的分析将提供新的见解的过程中，减轻问题所造成的复制压力，并有助于忠实的染色体分离。

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.