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 enter mitosis with unresolved repair intermediates or under-replicated DNA, where the physical connections between sister chromatids will manifest as microscopically visible chromatin- or ultrafine bridges that interfere with chromosome segregation. Recent findings suggest that there are several pathways that resolve these chromatin bridges, but the precise mechanism remains ill understood. Moreover, the machinery that contribute to the resolution of chromatin bridges remain to be identified.
We will combine quantitative mass spectrometry and microscopy based cell imaging techniques to identify and validate novel factors involved in the resolution of chromatin bridges. Using chromatin immunoprecipitation, we will isolate mitotic chromatin complexes and determine their composition by quantitative mass spectrometry. In collaboration with Holger Bastians (SP2), Matthias Dobbelstein (SP4) and Zuzana Storchová (SP8) we will characterize mitotic complexes in model systems with different types of replication stress. From the same complexes, we will analyze the bound DNA by next generation sequencing (in collaboration with the SP-Z) to determine sites where chromatin bridges form recurrently. Using microscopy (Zuzana Storchová, SP8), we will validate candidate proteins by their co-localization with known markers at distinct mitotic structures. The most promising candidates will be selected and analyzed further in order to precisely determine their function in the maintenance of genome stability. By acquiring a global multidimensional mitotic chromatin interactome through analysis of a range of different replication stress conditions we aim at identifying new classes of chromatin bridges and factors involved in their resolution.