| Maintenance of genomic stability during DNA replication involves the cooperation of many genes and processes. If DNA replication becomes dysfunctional, double strand breaks (DSBs) can arise which can lead to cellular senescence, apoptosis and even tumorigenesis. To combat the potentially harmful effects of faulty DNA replication, the cell has evolved DNA damage response (DDR) genes to repair DNA damage.;One such DDR gene, Brca2, functions in DSB repair through mediating homologous recombination (HR). Tumors that arise from mutation in Brca2 are deficient in HR and thus, rely on other DDR genes to counter any DNA damage that arises in the cell. One attractive way to design therapies to treat tumors that arise from specific DNA damage repair defects is to design synthetic lethality screens to determine if loss of a specific DDR gene can increase cellular death in these cancerous cells. Here, we have identified that loss of 7 different DDR genes are synthetic lethal with Brca2 mutation using murine embryonic fibroblasts. These genes (Rad52, DinB, Eme1, RPA, Chk1, Timeless, and Tipin) are involved in such processes as single-strand annealing, translesion synthesis, homologous recombination resolution, and intra-S phase checkpoint signaling. While our results could not be verified at the time, the identification of Timeless (Tim) and Tipin, which had relatively unknown function in mammals, piqued our interest and warranted further investigation into Tim's function during normal DNA replication.;In chapter III, our results demonstrate that Tim maintains genomic stability by preventing DSBs that create a dependence on HR-mediated mechanisms for repair during S phase. We show that Tim reduction increases DSB formation, Rad51/Rad52 foci formation, and HR as measured by sister chromatid exchange during DNA replication. These recombination events, created by Tim deficiency, were largely mediated through a Brca2/Rad51-dependent mechanism. These data, taken together indicated that Tim is pivotal for maintaining genomic stability during S phase. Thus, Tim reduction cooperates with Brca2-mediated HR repair to prevent loss of genomic integrity. |
