The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential RING E3 ligase that regulates cell cycle progression by polyubiquitinating substrates and targeting them for proteasomal degradation. Additionally, non-cell cycle roles of the APC/C have been described, and there is a growing interest in revealing the scope of APC/C activity in cellular functions. However, the dynamics of substrate ubiquitination by the APC/C are very complex and the identification of new substrates is very

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... ging provided the transient nature of substrate-APC/C interactions. Here, we describe a reliable bioinformatic tool to predict APC/C substrates that overcomes the technical challenges in identifying substrates and define a new role of APC/C activity in chromatin regulation (Chapter 2). APC/C polyubiquitinates its substrates by recruiting its substrates and simultaneously recruiting and activating its canonical E2s, UBE2C and UBE2S, in a sequential and independent manner. First, APC/C recruits and activates UBE2C~Ub to prime substrates (the addition of the first ubiquitin), and then it recruits and activates UBE2S~Ub to extend long ubiquitin chains. This interplay between APC/C, UBE2C, and UBE2S to polyubiquitinate substrates is often described as a unidirectional mechanism that portrays E2s as ubiquitin carriers. Interestingly, we show in the context of the APC/C that E2s can be more than ubiquitin carriers and this results in a multidirectional mechanism (Chapter 3). Via an allosteric positive feedback mechanism, UBE2S binds and activates the APC/C to stimulate UBE2C~Ub activity and enhance the efficiency of polyubiquitination. Overall, the findings in this thesis provide a new tool to identify potential APC/C substrates and broaden our understanding of the dynamics of substrate ubiquitination by the APC/C and its E2s and potentially by other E3-E2 systems.