Mechanistic studies of cellular oxidative stress response and DNA replication after DNA damage
Creators: name: Lu, Kevin Tsengchang
Publisher: name: No Publisher Supplied
Year: 2023

description: The protection of cellular genome against genotoxic and replication stressors requires the concerted effort of multitude of proteins with diverse functions. The transcription factor NRF2 upregulates oxidative stress response genes by binding to antioxidant response elements (ARE) during oxidative stress. In the absence of stress, NRF2 flux is regulated by KEAP1-dependent, proteasome-mediated degradation. KEAP1 regulates NRF2 by binding to a conserved "ETGE" and a “DLG” motif. The “ETGE” motif is not exclusive to KEAP1 and has also been identified in several other proteins, such as DPP3, which has been shown to bind KEAP1 and enhance NRF2 function upon overexpression. Here we demonstrate the interaction between endogenous DPP3 and endogenous KEAP1 occurs in a hydrogen peroxide inducible manner, and this interaction stabilizes the latter. Moreover, DPP3 is necessary for timely NRF2 induction and nuclear accumulation in the estrogen receptor (ER)-positive MCF7 breast cancer cells after ROS stress. Finally, we show that not only is DPP3 overexpressed in breast cancer, but also elevated levels of DPP3 mRNA correlate with increased NRF2 downstream gene expression and poor prognosis, particularly for ER-positive breast cancer. The regulation of NRF2 by DPP3 may provide potential transcriptional markers useful for prognosis and treatment for breast cancers.Cell Division Cycle 6 (CDC6) is an essential replication factor that mediates the assembly of the pre-replication complex (pre-RC) at the Origin Recognition Complex (ORC) during the G1 phase of the cell cycle. After replication initiation, CDC6 is phosphorylated by Cyclin Dependent Kinases (CDKs) and exported to the cytoplasm. CDC6 then remains cytoplasmic throughout G2. These spatial and temporal features of CDC6 help to ensure the faithful replication of the genome once and only once per cell cycle. Interestingly, we observed that ionizing irradiation (IR) and certain chemotherapeutic DNA damaging agents induce CDC6 nuclear accumulation during G2 in an ATR-Chk1-CDK pathway dependent manner. Moreover, we found that CDC6 serine 74 phosphorylation status is a major determinant of CDC6 localization not only before and but also after IR. Lastly, we also observed that depletion of CDC6 delays cell cycle recovery after DNA damage and causes aberrant mitotic entry leading to genome instability.Our lab recently reported that BRCA2 interacts with MCM10 at the replication fork and blocks PRIMPOL-mediated repriming of DNA synthesis downstream of DNA lesions, thereby suppressing the formation of single-stranded replication gaps. We showed that BRCA2 binds to a conserved coiled-coil (CC) motif at the N-terminus of MCM10; however, the domain/motif in BRCA2 that binds to MCM10 is unknown. Here, I have used Alpha-fold and a fluorescent based co-localization assay to predict the MCM10 binding site on BRCA2. The MCM10 α helix that constitutes the CC motif was used to “scan” BRCA2 amino acid sequences that was divided into 10 segments to identify the region(s) that possess highest affinity. Moreover, I also analyzed co-localization of full length mCherry-MCM10 and eight NLS-GFP-BRCA2 fragments to determine potential MCM10 binding sites in BRCA2 The results showed that two α helices in the BRCA2 “tower” domain and the helical domain are likely binding sites. This will be validated by experimental work in the future.