The Spindle Assembly Checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells entering anaphase by inhibiting the ubiquitination of cyclin B1 and securin by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator, Cdc20. It is unclear how the SAC inactivates Cdc20 but current models mostly involve Cdc20 forming a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC the APC/C ubiquitinates Cdc20 to target it for degradation. Thus, ubiquitination of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.

Treslin/TICRR (TopBP1-interacting, replication stimulating protein/TopBP1-interacting, checkpoint, and replication regulator), the human ortholog of the yeast Sld3 protein, is an essential DNA replication factor that is regulated by cyclin-dependent kinases and the DNA damage checkpoint. We identified MDM two binding protein (MTBP) as a factor that interacts with Treslin/TICRR throughout the cell cycle. We show that MTBP depletion by means of small interfering RNA inhibits DNA replication by preventing assembly of the CMG (Cdc45-MCM-GINS) holohelicase during origin firing. Although MTBP has been implicated in the function of the p53 tumor suppressor, we found MTBP is required for DNA replication irrespective of a cell's p53 status. We propose that MTBP acts with Treslin/TICRR to integrate signals from cell cycle and DNA damage response pathways to control the initiation of DNA replication in human cells.

Summary DNA replication in eukaryotic cells initiates from multiple replication origins that are distributed throughout the genome. Coordinating the usage of these origins is crucial to ensure complete and timely replication of the entire genome precisely once in each cell cycle. Replication origins fire according to a cell-type-specific temporal programme, which is established in the G1 phase of each cell cycle. In response to conditions causing the slowing or stalling of DNA replication forks, the programme of origin firing is altered in two contrasting ways, depending on chromosomal context. First, inactive or 'dormant' replication origins in the vicinity of the stalled replication fork become activated and, second, the S phase checkpoint induces a global shutdown of further origin firing throughout the genome. Here, we review our current understanding on the role of dormant origins and the S phase checkpoint in the rescue of stalled forks and the completion of DNA replication in the presence of replicative stress.