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

点击翻译按钮获取中文摘要

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.