Abstract
Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes – may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.
Highlights
Precise replication of genomic DNA before each cell division is essential for maintaining the integrity of genetic information in proliferating cells and through succession of generations
The results of our mass spectrometry analysis showed that after excluding the common contaminants and background interactors, the most abundant specific partners of MCM3 were the other subunits of MCM2-7 complex, Keap[1], and MCM-BP (Supplementary Fig. S1)
Quantification of Keap1 - MCM3 proximity specific proximity ligation analysis (PLA) dots confirmed that the interaction between these two proteins takes place in both nuclear and cytoplasmic compartments, with some preferential bias towards cytoplasm (Fig. 1d), while the parallel immunofluorescence analysis expectedly revealed that MCM3 is mostly nuclear and Keap[1] mostly cytoplasmic in these cells (Fig. 1e)
Summary
Precise replication of genomic DNA before each cell division is essential for maintaining the integrity of genetic information in proliferating cells and through succession of generations. We propose that such competitive binding mechanism may have enabled the Keap1-Nrf[2] pathway to adjust to the status of replication machinery in the cell; the levels of MCM3 competitor, or its availability for Keap[1] binding, serving as an indicator of such status This prototype MCM3 dependent modulation mechanism of Keap[1] controlled cellular functions might have further evolved to incorporate similar competitive binding dependent sensory feedback from other proteins and cellular processes[30,31], possibly enabling precise tuning of the Keap[1] controlled regulatory network in response to a wide range of cellular conditions. Our data suggest about possible involvement of MCM7, another subunit of MCM2-7 complex, and MCM-BP, a protein that can dissociate and unload MCM2-7 complexes from chromatin[32,33,34], in the Keap1-MCM3 interaction related regulatory pathways
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
