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.

Figure 1

Depletion of CTC1 or STN1 causes genomic instability in HeLa 1.2.11 cells. (A) Western blot showing knockdown of STN1 (42 kDa) in shSTN1 clones and re-expression of an sh-resistant Flag-STN1 (shSTN1-7 Res). Loading control is α-Actinin (100 kDa). Lanes 1–3 contain 25 μg of protein and lane 4 contains 10 μg. Numbers below gel indicate the level of STN1 relative to non-target control (shNT) after normalization to α-Actinin. (B) RT–qPCR of STN1 and CTC1 mRNA in different clones. Levels are relative to shNT with normalization to GAPDH (mean±s.e.m., n=3 independent experiments). (C, D) Anaphase bridges observed after release of control, shSTN1 or shCTC1 clones from nocadozole block (mean±s.e.m., n⩾3 independent experiments). NT, non-target; WT, wild type.

Figure 2

CTC1 or STN1 depletion cause multi-telomeric signals (MTS). (A, C) Telomere FISH of HeLa 1.2.11 shCTC1 or shSTN1 clones (A) or U2OS shSTN1 clone (C) showing examples of MTS (white arrows). Green, FITC-telomere probe; blue, DAPI. (B) Quantification of MTS in HeLa 1.2.11 cell lines. Metaphase spreads were made from cells grown±0.25 μg/ml aphidicolin for 16 h prior to the addition of colchicine or colcemid (mean±s.e.m., n⩾3 independent experiments). (D) Quantification of MTS from a single experiment with a U2OS STN1 knockdown clone. NT, non-target; WT, wild type.

Figure 3

STN1 depletion delays telomere replication but does not affect the rate of bulk genomic DNA replication. (A) Experimental timeline. HeLa 1.2.11 cells were released from a double-thymidine block into S-phase and incubated with BrdU or EdU for consecutive 1.5 h intervals. (B) Rates of bulk genomic DNA replication were determined by EdU uptake. Graph shows EdU incorporated at consecutive time periods (Mean EdU staining X % EdU-positive cells). (C–E) Rates of telomere replication throughout S-phase. (C) BrdU-labelled DNA from 4.5 and 6 h time points was subject to CsCl sedimentation to separate leading and lagging strand telomeres. Telomeric DNA from each gradient fraction was quantified by slot blot hybridization. (D) Per cent of newly replicated leading strand telomere signals relative to the total telomere signal for each time period throughout S-phase. (E) Examples of slot blot used to obtain data in (C) and (D). Data are representative of three independent experiments. NT, non-target.

Figure 4

Co-depletion of TRF1 and STN1 causes an additive increase in MTS. (A) Relative level of TRF1 mRNA 48 h after siRNA transfection as measured by RT–qPCR with normalization to GAPDH (mean±s.e.m., n=3 independent experiments). (B) Quantification of MTS (mean±s.e.m., n=3 independent experiments). Dashed line indicates the background level of MTS. NT, non-target.

Figure 5

Replication restart after HU treatment is inhibited by STN1 depletion. (A–C) Cells were treated for 2 h with 2 mM HU and released into medium containing EdU for 30 min. (A) EdU incorporation by HeLa 1.2.11 clones after release from HU. Blue, DAPI; green, EdU. (B) Quantification of the levels of EdU uptake after release from HU, as measured by mean fluorescence intensity (mean±s.e.m., n⩾3 independent experiments). Each bar indicates the total number of nuclei above or below the AFU given below. AFU, arbitrary fluorescence units. Nuclei below 10 AFU are considered as EdU negative, those above 10 AFU are EdU positive. (C) Average AFU values of all nuclei following HU removal for both HeLa1.2.11 knockdown clones and pools of U2OS knockdown cells (mean±s.e.m., n⩾3 independent experiments). NT, non-target.

Figure 6

Rapid disappearance of RPA foci after release from HU. shNT (top row of each panel) or shSTN1-7 (bottom row of each panel) cells were incubated with EdU to label S-phase cells, then treated with 2 mM HU for 2 h and fixed without release (top left), released for 8 min prior to fixation (top right) or 12 minutes prior to fixation (bottom left). No HU treatment is shown (bottom right) to represent RPA foci that occur naturally during replication. Images are shown from a single experiment and are representative of three independent experiments. Red, RPA; green, EdU; blue, DAPI. NT, non-target.

Figure 7

STN1 depletion leads to decreased new origin firing following release from HU-induced fork stalling. (A) Schematic of experimental approach and types of DNA fibres scored. HeLa 1.2.11 cells were pulse labelled with IdU and CldU, as indicated, to label individual replication forks. DNA fibre spreading was then performed (see Materials and methods) and IdU/CldU visualized by immunofluorescent labelling. Images indicate the different replication events observed. (B) Representative images of DNA fibres. Red, IdU; green, CldU. (C) Graphical representation of the percentage of red-only (stalls or terminations) or green-only (new origins fired during CldU pulse) tracks (mean±s.e.m., n=3 independent experiments). (D) Total number of tracks scored. In parenthesis is the percentage of the total number of tracks scored. NT, non-target; WT, wild type.