During DNA replication, lesions generated by UV exposure result in replication fork stalling. To synthesize DNA past these lesions, replicative polymerases (Pol?) must be exchanged with special polymerases (Pol?), which can incorporate nucleotides opposite of lesions. This process is named Translesion Synthesis (TLS). The mechanism of polymerase switching remains unclear. Monoubiquitination of Proliferating Cell Nuclear Antigen (PCNA) is required for this process to occur efficiently. Our lab has previously identified a protein named Spartan that binds to ubiquitinated PCNA and promotes the recruitment of Pol? to DNA lesions. It is currently unclear how Spartan mediates the polymerase switch. My preliminary data suggests that during UV exposure Spartan regulates the stability of an accessory subunit of the replicative polymerase, POLD4. The importance of TLS in cancer is exemplified by the autosomal recessive disease Xeroderma Pigmentosum Variant (XPV) in which Pol? is functionally inactive. XPV patients have an increased sensitivity to UV exposure that results in a dramatic increased susceptibility to skin cancer. Searching the genomics online database Sanger COSMIC, revealed that 10% of breast cancer tissues tested had SPRTN/C1orf124 amplifications and 30% had overexpression. This suggests that Spartan dysregulation might influence breast cancer development by enabling cells to handle replication stress that accompanies genome instability, a hallmark of cancer.
The specific aims of this proposal are as follows: (1) To determine whether Spartan regulates the Pol?-to-Pol? switch by promoting POLD4 degradation; (2) to elucidate how Spartan regulates TLS through its SprT domain; (3) to determine whether amplification of SPRTN in breast cancer offers an opportunity for therapy. To address these aims, Spartan mutants that disrupt the various functional domains will be tested for their ability to promote POLD4 dissociation from PCNA, and Pol? association with PCNA. These interactions and co-localizations will be assessed by co-IP and Immunofluorescence, respectively. In vitro assays will be used to determine whether the SprT domain harbors protease activity and cleaves POLD4. Also, a Spartan inducible stable cell line will be generated. This will be used to reveal the impact of Spartan overexpression on the tolerance of untransformed mammary epithelial cells to extrinsic DNA damage or intrinsic genomic instability. The influence of shRNA mediated Spartan knock-down on breast cancer cell viability and proliferation will also be assessed. The short-term contribution of the proposed research will be the illumination of the mechanism of Spartan in TLS, a cancer relevant pathway. The long-term contribution could be the development of new drugs for eliminating cancer.
The proposed research will illuminate the potential impact of a recently identified protein dubbed Spartan, in the survival of breast cancer cells and its mechanism of action. The resulting information may provide the basis for new cancer therapeutics that target Spartan and its associated regulators. This could be the Achilles' heel of breast cancer, and result in enhanced treatment regimens for suffers of the disease.
