Author ORCID Identifier
Date of Graduation
Genetics and Epigenetics
Doctor of Philosophy (PhD)
The human genome is under constant threat from sources of damage and stress. Improper resolution of DNA damage lesions can lead to mutations, oncogene activation, and genomic instability. Difficult-to-replicate-loci present barriers to DNA replication that, when not properly resolved, lead to replication fork stalling and collapse and genomic instability.
DNA damage and replication stress trigger signaling cascades potentiated by multiple types of post-translational modifications, including SUMOylation. Through proteomic analysis of proteins involved in SUMOylation following DNA damage, our lab identified an uncharacterized protein that we named New Player in SUMO-dependent DNA damage repair 4 (NPSD4). Through an additional proteomic screen, we found that NPSD4 interacts with DNA repair and replication proteins, suggesting a role of NPSD4 in DNA repair and replication.
In this study, I investigated the SUMO-mediated function of NPSD4 and demonstrated that NPSD4 plays a critical role in protecting stalled replication forks in response to replication stress. I showed that NPSD4 interacts with SUMO2/3 via two SUMO interacting motifs (SIMs). In addition, I found that NPSD4 is a nuclear protein and can localize to the SUMOylated promyelocytic leukemia nuclear bodies (PML NBs) in a SIM-dependent manner. With collaboration in the lab, we show that NPSD4 interacts with and regulates the protein levels of BLM helicase, an important regulator
of recombination which also can localize to PML NBs. The interaction of NPSD4 with BLM and the regulation of BLM levels by NPSD4 are SIM-dependent. Importantly, my work shows that the increased BLM levels in NPSD4 knockout cells lead to stalled replication fork degradation. I also found that the fork degradation defect in NPSD4-deficient cells is dependent on the DNA2 nuclease and proteins that promote fork reversal. Thus, my findings indicate that NPSD4 protects stalled replication forks through limiting BLM levels, inhibiting aberrant resection of reversed stalled forks by BLM/DNA2. These results identify a novel fork protection mechanism. The study of NPSD4 will provide novel insights into the mechanisms of SUMO-regulated replication stress signaling and the maintenance of genomic stability.
DNA replication, replication stress, DNA damage, SUMO, BLM helicase, stalled fork degradation
Available for download on Thursday, July 13, 2023