Thesis Format
Alternative Format
Degree
Master of Science
Program
Biochemistry
Supervisor
Ling, Hong
Abstract
Translesion synthesis (TLS) bypasses DNA lesions by using error-prone DNA polymerases recruited by mono-ubiquitinated PCNA (Ub-PCNA). To reduce the mutagenic consequences of TLS, the USP1/UAF1 complex removes ubiquitin from PCNA, terminating the TLS process. This study investigated the role of ATAD5 in the PCNA de-ubiquitination (DUB). We demonstrated that ATAD5 binds USP1/UAF1 and Ub-PCNA simultaneously as a molecular adaptor, connecting the DUB enzyme and the substrate to promote DUB reactions. The SIM domain of ATAD5 binds the SUMO-like domains (SLD1 and SLD2) of UAF1 and has canonical interactions with SLD2. Furthermore, we found that the disordered linker of ATAD5 contributes partially to its regulatory activity. Our structural studies of UAF1/SIM chimeras yielded crystals diffracting to 7.6 Å resolution for SIM-SLD interface analysis. These findings provide new insights into the molecular mechanisms of TLS regulation, underscoring the potential of ATAD5 as a therapeutic target for inducing genomic instability in cancer cells.
Summary for Lay Audience
All cells require an accurate copy of the genetic blueprint to live and proliferate. The genetic code is duplicated using DNA-replicating polymerase proteins that proofread the code before passing it on to the next generation. Cells are constantly exposed to stressors that can alter or damage DNA, leading to developmental abnormalities or cancers. DNA lesions may also stall DNA replication and ultimately kill the afflicted cells. To avoid these outcomes, cells bypass the damage sites through translesion synthesis (TLS). This process is promoted by the addition of a ubiquitin molecule to the DNA-clamping protein, PCNA, that recruits specialized polymerases to finish DNA replication at the cost of potential mutations. However, it is critical to have a shut-off mechanism for this error-prone replication is no longer necessary. To return to normal replication, ubiquitin is cleaved from PCNA by the USP1/UAF1 enzyme complex. In the presence of ATAD5 protein, ubiquitin cleavage is further enhanced, indicating a regulatory role of ATAD5 in terminating TLS. We hypothesize that ATAD5 achieves this by tethering PCNA and USP1/UAF1, allowing them to come in proximity for an increased chance of ubiquitin cleavage. Previous work has demonstrated the binding interaction between ATAD5 and PCNA, but the interaction between ATAD5 and USP1/UAF1 remains unclear. Using functional assays and crystallization, we prove that the SIM domain of ATAD5 binds to the SLD2 region on UAF1 through a classical SIM-SUMO interaction, and with the SLD1 region on UAF1 in a non-classical interaction. We have also successfully generated protein crystals that will ultimately allow the visualization of the ATAD5-UAF1 interaction at atomic resolutions. These results will increase our understanding of the TLS pathway and may lead to the development of new cancer therapeutics.
Recommended Citation
Truong, Vo Ha Uyen, "ATAD5 Dual-Domain Interaction with UAF1 Enhances USP1-mediated PCNA De-ubiquitination in the TLS Pathway" (2024). Electronic Thesis and Dissertation Repository. 10643.
https://ir.lib.uwo.ca/etd/10643
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