The Mechanisms and Consequences of DNA Re-replication

DNA replication is a key event during cellular proliferation. In order to maintain genetic stability, cells have evolved different mechanisms to ensure the precise duplication of chromosomes and prevent DNA re-replication. The regulation of DNA replication initiation is critical for preventing re-replication. In mammalian cells, stabilization and activation of the replication initiator Cdt1 leads to DNA re-replication. In this dissertation, I examine the consequences of the re-replication. The single stranded DNA (ssDNA) initially generated during re-replication activates an ATR/Chk1 mediated pathway and arrests cells in G2/M phase. The checkpoint is essential for the accumulation of re-replicated cells, which can further activate ATM/Chk2, p53, and apoptosis. Our study suggests that cells can have a chance to repair relatively minor DNA damage caused by microscopic re-replication, and only induce apoptosis through the later acquisition of double strand breaks and activation of Chk2 and p53 when re-replication persists. We also identified HDAC6 deacetylase as a new player in re-replication induced checkpoint pathways, presumably by regulating Chk1 protein level and phosphorylation, directly or indirectly. MLN4924, a new anti-cancer drug, stabilizes Cdt1 and causes re-replication in a variety of human cancer cells. Transient exposure of MLN4924 is sufficient to induce rereplication, which activates checkpoint pathways, apoptosis, and senescence, contributing to the anti-proliferative effect of MLN4924 in cancer therapy. Intriguingly, unlike other III DNA damaging agents used for chemotherapy, p53-negative cells remain susceptible to MLN4924 induced cell death, suggesting an important clinical application. Although the consequences of re-replication, such as apoptosis and senescence, can be used for killing cancer cells, re-replication itself may lead to gene amplification and tumorigenesis. However, we found no evidence of this deleterious effect in normal breast epithelial cells or breast cancer cells, most likely because re-replication-induced checkpoint, apoptosis, and senescence acted as insurmountable barriers to tumorigenesis.

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