DNA replication in eukaryotic cells is tightly controlled so that the genome is replicated once and only once per cell cycle. Disruption of DNA replication control due to impaired replication licensing mechanisms causes DNA rereplication, which often leads to genome instability, contributing to tumorigenesis. In support of this, deregulated overexpression of the licensing factor Cdt1 is associated with a large panel of tumors. Furthermore, a number of oncogenes are found to induce DNA rereplication at the early stage of cancer development. Thus, DNA rereplication is a driving force for tumorigenesis. DNA double-strand breaks (DSBs) are frequently formed during rereplication, but the mechanisms underlying the repair of rereplication-induced DSBs to maintain genome integrity are still elusive. Since DNA rereplication produces extra copies of DNA segments and generates multiple DSBs, the associated repair process is expected to be much more complex than that for a general single DSB. In this study, we will investigate the detailed mechanisms of how rereplicated DNA is removed, and how DSBs generated at rereplication forks are repaired by using our newly established novel EGFP-based DSB repair substrates. We will also study the consequences for genome instability that rereplication would cause, such as chromosomal lesions and gene amplification. Since DNA rereplication is an integral aspect of tumorigenesis, our proposed study will provide insights into new mechanisms associated with tumor initiation and development, and will also shed light on developing strategies for cancer diagnosis and treatment in the future.