Human CST promotes telomere duplex replication and general replication restart after fork stalling

Abstract

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing.

Human TEN1 Maintains Telomere Integrity and Functions in Genome-wide Replication Restart

“…Elsewhere in the genome, STN1 and CTC1 participate in the resolution of replication stress (10,29). Depletion of either subunit leads to formation of anaphase bridges, and STN1 depletion results in reduced replication restart after hydroxyurea (HU)-induced fork stalling (10,11,29). Consistent with this genome-wide role in replication, CTC1 and STN1 were originally identified as a pol ␣ accessory factor (AAF) that stimulates template binding and enzyme processivity (31,32).…”

“…Primers for TEN1 amplification, 5Ј-GGCCAAGTTCCT-GATGGG and 5Ј-CAGTGTTACTCTGGACTGAATCAT, were designed to avoid amplification of a TEN1 pseudogene. GAPDH and STN1 primers were as described (10). Reactions were performed at least in duplicate using GAPDH as the endogenous control.…”

“…At the telomere, STN1 and CTC1 function both in telomere duplex replication and C-strand fill-in (10 -12, 14, 29, 30). Elsewhere in the genome, STN1 and CTC1 participate in the resolution of replication stress (10,29). Depletion of either subunit leads to formation of anaphase bridges, and STN1 depletion results in reduced replication restart after hydroxyurea (HU)-induced fork stalling (10,11,29).…”

“…The mutant allele was cloned into the pMSCV-IThy1-1 vector. Viral production, infection, sorting of Thy1-1-expressing cells, and production of clones were as described (10).…”

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

“…CST: DNA Pol‐α‐mediated C‐strand fill‐in is absolutely required for telomere length maintenance, as telomerase by itself is insufficient to generate the proper duplex telomere (Feng et al., 2017; Gu et al., 2012). Defects in telomere replication due to disruption of the CST complex lead to replication fork stalling, as telomeres can adopt secondary structures that are difficult to replicate (Gu et al., 2012; Stewart et al., 2012). Stalled replication forks and the failure of stalled fork restart at telomeres initiate aberrant homologous recombination events that in part account for the catastrophic loss of total telomeric DNA observed in mouse cells devoid of CTC1 (Gu et al., 2012), or the activation of a DDR in mammalian cells (Wang et al., 2012).…”