Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association

Abstract

Human CTC1-STN1-TEN1 (CST) is an RPA-like single-stranded DNA-binding protein that interacts with DNA polymerase α-primase (pol α) and functions in telomere replication. Previous studies suggest that CST also promotes replication restart after fork stalling. However, the precise role of CST in genome-wide replication remains unclear. In this study, we sought to understand whether CST alters origin licensing and activation. Replication origins are licensed by loading of the minichromosome maintenance 2–7 (MCM) complex in G1 followed by replisome assembly and origin firing in S-phase. We find that CST directly interacts with the MCM complex and disrupts binding of CDT1 to MCM, leading to decreased origin licensing. We also show that CST enhances replisome assembly by promoting AND-1/pol α chromatin association. Moreover, these interactions are not dependent on exogenous replication stress, suggesting that CST acts as a specialized replication factor during normal replication. Overall, our findings implicate CST as a novel regulator of origin licensing and replisome assembly/fork progression through interactions with MCM, AND-1, and pol α.

Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

“…Unlike yeast, mammalian CST is involved in several distinct DNA transactions in the cell, including telomere maintenance, replication rescue, dormant origin firing, origin licensing and DSB repair (23,(25)(26)(27)29,30,34). However, it has been difficult to identify the contribution of telomere versus non-telomere CST functions on genome stability and cell cycle progression.…”

“…In a similar manner, CST could stabilize TopBP1 by recruiting it to the chromatin. As to the mechanism of CST recruitment to stalled forks, we recently showed that CST interacts with the MCM2-7 helicase (25). Thus, CST could be recruited to stalled forks by MCM2-7 and then recruits TopBP1 to prevent degradation, stimulating ATR-mediated CHK1 phosphorylation.…”

“…In agreement with previous findings, we observed decreased cell proliferation starting around 6 days after conditional CTC1 deletion, which became more severe at 10-12 days in culture ( Figure 1B). We expected that the growth delay was caused by an intra-S-phase checkpoint triggered by replication issues previously observed after depletion of CST subunits (25,29,30,37). However, while cell cycle analysis revealed a substantial decrease in the number of Sphase cells following CTC1 deletion, the cells within S-phase retained similar levels of DNA synthesis (EdU intensity per cell) compared to controls ( Figure 1C-D), suggesting that replication is not inhibited in the CTC1 -/cells.…”

“…Cell pellets were lysed, sonicated and nuclease-treated as previously described (25). The supernatant was collected and protein concentration measured by BCA assay.…”

“…Like RPA, CST has multiple oligosaccharide-oligonucleotide binding folds (OBfolds) that allow dynamic binding to ssDNA. CST plays a critical role in telomere replication and has recently been shown to function in genome-wide replication and DSB repair (24)(25)(26)(27)(28)(29)(30). At telomeres, CST promotes telomerase dissociation following telomere extension and is then required to convert the G-rich ssDNA overhang (G-overhang) to duplex DNA through a process known as C-strand fill-in (31,32).…”