Analyses of the genome stabilising function of the tumor suppressor BRCA1 in mitosis.

点击翻译按钮获取中文摘要

Human cancer cells are characterized by numerical and structural chromosome aberrations that are caused by whole chromosome instability (W-CIN) and structural chromosome instability (S-CIN), respectively. BRCA1 represents an important tumor suppressor known to be involved in the regulation of DNA replication and DNA repair. Consequently, loss of BRCA1 in cancer cells causes S-CIN. Our most recent work has revealed another key role for BRCA1 and for several BRCA1-regulating proteins in mitosis required for the suppression of W-CIN. In fact, loss of the mitotic function of BRCA1 results in abnormally increased microtubule plus end assembly rates causing whole chromosome missegregation during anaphase. Whether the mitotic and the interphase functions of BRCA1 are functionally coupled and how BRCA1 fulfills its role during mitosis are unresolved questions that will be addressed within the time frame of the proposed research project.In the first part of the working program we will investigate whether the mitotic and interphase functions of BRCA1 are functionally linked. For this, we will establish BRCA1-degron systems in human somatic cells by CRISPR/Cas9 technology that allow the rapid depletion of endogenous BRCA1 at specific time points during interphase and in mitosis. In this way we aim to examine the different functions of BRCA1 after acute loss of BRCA1 in defined cell cycle phases and to explore their relative contributions to the suppression of S-CIN and W-CIN. The goal of these investigations is to elucidate whether and how S-CIN and W-CIN are linked upon loss of BRCA1.Since our work demonstrated that the mitotic function of BRCA1 involves its ubiquitin ligase activity it is pivotal to identify physiologically relevant ubiquitin acceptor proteins for the BRCA1 ubiquitin ligase in order to understand how BRCA1 fulfills its role during mitosis. Based on our preliminary work to identify ubiquitinated proteins we will use mass spectrometry to identify proteins that are ubiquitinated by the BRCA1 ubiquitin ligase during mitosis. Those candidate proteins will then be validated as BRCA1 targets in vitro and in vivo and we will investigate the role of the ubiquitin modification for the regulation of mitotic microtubule assembly and for accurate mitotic chromosome segregation. Thus, the proposed project with its two complementary working packages aims to unravel how BRCA1 contributes to genome stabilization in particular during mitosis.