ChIP-MS protocol

Version 1

Our Research Unit addresses an important question on the origin of chromosome instability (CIN), which causes structural as well as numerical chromosome aberrations. Importantly, CIN and increased levels of chromosome aberrations are closely associated with many human diseases including cancer, neurodegenerative diseases and age-related syndromes and can act as key drivers for disease development and progression. An important condition that causes structural chromosome instability (S-CIN) leading ...

Replication stress is a key driver of genomic instability giving rise to human disease, for instance cancer or developmental disorders. It is caused by a variety of defects such as lesions blocking replication (e.g. DNA interstrand crosslinks) or processes that lead to the exhaustion of DNA precursors. This generates under-replicated DNA and replication intermediates that can be rescued by repair processes requiring homologous recombination between sister chromatids. Often, cells exposed to ...

Replication stress is one of the major causes for chromosomal instability which is a hallmark of cancer. Incomplete repair can lead to severe mitotic defects and ultra-fine anaphase bridges (UFBs) are a typical consequence. So far, five different classes of UFBs have been discribed: fragile sites-UFBs, centromeric-UFBs, telomeric-UFBs, homologous recombination-UFBs and rDNA-UFBs, which can be distinguished by their location and the way how they arise. Although previous studies have already proposed ...

FANCD2 ChIP-MS experiments in refined synchronized HCT116 cells with different types of replication stress are performed to identify additional proteins involved in the resolution of UFBs. Through reciprocal ChIP-MS and colocalization analysis by immunofluorescence experiments observed interactions will be validated. In collaboration with SP-Z promising candidates will be prioritized and subsequently tested for their contribution to the resolution of UFBs and chromosome segregation. In collaboration ...

CHIP-MS was performed with HeLa cells carrying a bacterial artifical chromosome for the expression of GFP-tagged PICH under it's endogenous promotor. Cells were treated with drugs to induce different types of ultra-fine bridges (UFBs). Treatments included: a) 24h exposure to 0.1 uM Aphidicolin, b) 24 hours exposure to 120 ng/ml mitomycin C, c) 6.5 hours incubation in 0.05 ug/ml nocodazol followed by a release into 0.1 uM ICRF-193 topoisomerase inhibitor.