In the last cycle of this project we discovered how easy it is for mammalian cells to bypass the regulatory pathways that ensure that origins of replication are licensed only once per cell-cycle and that the chromosomes are replicated once and only once per cell-cycle. Different manipulations that increased the ratio of the replication initiator, Cdt1, relative to its inhibitor, geminin, led to re-replication. Intriguingly, the extensive re-replication was accompanied by DNA damage and activation of checkpoint pathways that led to cell-cycle arrest and eventually cell-death. The checkpoint pathways used several genes whose mutations predispose individuals to genomic instability and cancer: p53, BrCa1 and the Fanconi Anemia proteins. These results lead us to the hypothesis that will be tested in the first Aim: small degrees of re- replication that are tolerated and are compatible with cancer cell viability lead to the deletions and gene amplifications that are the hallmark of cancer cell chromosomes. This will be the first model linking disorders in replication initiation in mammalian cells to chromosomal structural variation and the genomic instability of cancer. A first-in-its-class new drug has been developed by a pharmaceutical company and will be given to us. This drug, MLN4924, inhibits the ubiquitin ligases that help degrade Cdt1 in S phase, and as a result stabilizes Cdt1 and causes extensive re-replication, DNA damage and cytotoxicity. Aim 2 will test the hypothesis that the cytotoxic effect of this drug is through the re-replication and activation of checkpoint pathways. If this hypothesis is true, we will provide a rationale for the development of other anti- cancer drugs that produce extensive re-replication and successfully predict whether the clinical trial of MLN4924 will benefit from a measurement of the status of p53, BrCa1 or Fanconi Anemia proteins in the tumors being treated. Although we have discovered two independent ubiquitin ligases that promote the polyubiquitinylation of Cdt1 and its degradation, preliminary results indicate that a third ubiquitin ligase of an entirely different class from the ones already implicated, has a critical role in destabilizing Cdt1. In the third Aim we will test the hypothesis that inhibition of this third ubiquitin ligases will also cause re-replication and cytotoxicity, determine the mechanism by which this ligase destabilizes Cdt1 and assess whether inhibition of this ligase will synergize with MLN4924 in cancer chemotherapy.