Cancer cells rely on DNA damage tolerance pathways to cope with intrinsic oncogenic stresses and evade DNA-damaging environmental and therapeutic agents. However, the mechanisms by which neoplastic cells hijack tightly controlled DNA damage tolerance-signaling cascades to promote mutagenesis and chemoresistance are not understood. Thus, limitations in our knowledge of DNA damage tolerance and mutagenesis impede effective prevention and treatment of cancer. We have discovered two unique regulators of RAD18 and replication-associated DNA damage tolerance that are overexpressed in cancer: RNF168 (an apical mediator of double strand break signaling) and MAGEA4 (a cancer cell-specific protein with no known function). RNF168 is mutated in human RIDDLE syndrome, a disease characterized by severe immunodeficiency, developmental defects, radiosensitivity and a predisposition to cancer. We show here that RNF168 is a novel component of the RAD18 complex, facilitating its recruitment to stalled replication forks and promoting damage tolerance following replication stress. We have also identified the cancer/testis antigen (CTA) MAGEA4 as a stabilizing binding partner of RAD18 that promotes trans-lesion DNA synthesis. Thus, the findings in this thesis offer neomorphic cancer cell-specific roles for regulators of DNA damage tolerance. Identification of mechanisms of DNA damage tolerance that drive carcinogenesis and confer chemoresistance will allow for the development of more effective cancer treatment regimens. CTAs are absent from normal somatic cells but aberrantly overexpressed in many cancers. Interestingly, CTAs have been correlated with chemotherapeutic resistance and poor prognostic outcomes, though their contributions to carcinogenesis are not understood. We have found that depletion of several CTAs (MAGEA4, MAGEA10, or HORMAD1) sensitizes non-small cell lung cancer (NSCLC) cells to DNA-damaging therapies. These studies identify novel mechanisms by which NSCLC cells aberrantly overexpress germ cell proteins to alter genome maintenance, offering a cancer cell-specific mechanism by which neoplastic cells acquire chemoresistance and evade therapy. Accordingly, these CTAs are promising therapeutic targets whose inhibition should be innocuous to normal somatic cells while greatly sensitizing cancer cells to existing DNA damaging chemotherapeutic agents.