The anaphase promoting complex/ cyclosome (APC/C) is an E3 ubiquitin ligase that controls the cell division cycle by targeting main cell cycle regulators for proteasomal degradation, thereby ensuring error-free cell division and safeguarding genome stability. Its foremost activity is during cell division, or mitosis, when two sets of sister chromatids are equally divided over two newly formed daughter cells. Intriguingly, APC/C substrates that are degraded at the metaphase-to-anaphase transition have binding partners, which are not degraded. It remains a mystery how the APC/C controls degradation of the substrates and leaves the binding partners undisturbed. My objective is to clarify the molecular mechanism of substrate-binding partner disengagement, and determine the impact of disengagement on substrate degradation, to ensure controlled sister chromatid separation and genome integrity. First, I propose to identify the precise timing of disengagement during the process of ubiquitination, at the molecular level: this will give fundamental insight into disengagement control (Objective 1). Next, I will study ubiquitination at the proteomics level, by unraveling how Lysine-choice, and ubiquitin chain topology affect disengagement (Objective 2). Finally, I will combine conventional molecular biology methods with advanced microscopy techniques to investigate the importance of controlled substrate-binding partner disengagement for substrate degradation and genome stability (Objective 3). I will employ a multi-disciplinary approach, combining molecular biology, proteomics, and in vivo cell biology approaches to resolve this fundamental biological question. The identified mechanism may provide insights into ternary complex formation of the APC/C and its substrates, which will enable translation to develop targeted-protein-degradation drugs.