Tus is bound to TerB sites integrated in human cells

(A) Schematic of linearized plasmid (pWB15) integrated into MCF7 cells (MCF7 5C-TerB) with two cassettes containing 5 tandem TerB sequences in the non-permissive orientation (black arrows). Black half-arrow heads depict PCR products expected from stated primer pairs used in E. (B) ChIP with His antibody on MCF7 5C-TerB cells transfected with either a vector control (VC) or HA-Tus-His expression plasmids. ChIP-qPCR were conducted using the indicated primer pairs. Data show the percentage of input (n=3). (C) Schematic of PLA to visualize Tus bound to TerB sites using HA and His antibodies. (D) Representative images of the PLA foci across stated conditions. Bars, 10 μm (E) Percentage of cells with 1-2 PLA foci per nucleus. (n=3, ≥150 cells per experiment).

Replication fork pauses at the Ter sequence in the presence of the Tus protein

(A) Schematic of pulse labelling of MCF7 5C-TerB cells with Tus protein expression for 3 days. (B) Locus map of a 200 kb SfiI segment from MCF7 5C-TerB Chromosome 12 with the integrated plasmid (pWB15) DNA (orange). A 40 kb FISH probe made from fosmid WI2-1478M20 and a 7.5 kb FISH probe made from plasmid pWB15 are shown in blue. (C-D) Top: locus map of the DNA segment containing Ter sequence with the location of the FISH probes. Bottom: photomicrographs of labeled DNA molecules from MCF7 5C-TerB, Tus not expressed (C) and MCF7 5C-TerB, Tus expressed (D). Yellow arrows indicate the position of replication forks at the transition of labeling from IdU to CldU incorporation showing replication fork direction. Molecules are arranged in the following order: forks progressing from 3’ to 5’, forks progressing from 5’ to 3’, termination events, and initiation events. Replication forks (Yellow Arrows) in the white oval are all at the same location (at the TerB sequence) on molecules from different cells indicating that replication forks are pausing at this TerB sequence. (E-F) Percentage of molecules with replication forks at each 5 Kb interval in the 200 Kb SfiI segment containing TerB sequence, quantified from molecules in MCF7 5C-TerB (E) Tus not expressed and (F) Tus expressed. Percentage of molecules with replication forks progressing 3’ to 5’ (< blue) and 5’ to 3’ (> orange) are shown. In the cells expressing Tus, a high percentage of molecules contain replication forks pausing in both directions in the 5 Kb interval which contains Ter sequences (black arrow (F), white oval (D)). (G) Schematic similar to Fig1A with progression of the endogenous origins of replication within the chromosome 12 shown (green arrows). (H) ChIP using MCM3 antibody on MCF7 5C-TerB cells transfected with VC or HA-Tus-His plasmids. ChIP-qPCR were conducted using the indicated primer pairs. Data shows the fold enrichment relative to the IgG controls (n=3).

γH2AX is enriched at TerB sites after Tus expression

(A) MCF7 5C-TerB cells transfected with GFP or GFP-Tus, γH2AX and IgG antibodies were used to immuno-precipitate proteins and analysed by immunoblotting with indicated antibodies. (B) Schematic of PLA to visualize HA-Tus bound to TerB in the proximity of γH2AX sites using HA and γH2AX antibodies (C) Representative images of the PLA foci across stated conditions. Bars, 10 μm (D) Percentage of cells with 1-2 PLA foci per nucleus. (n=3, ≥150 cells per experiment). (E) Schematic of MCF7 5C-TerB depicting the positions of the primer pairs with respect to the integrated TerB locus. (F) γH2AX levels along the integrated TerB plasmid were analyzed by ChIP-qPCR in MCF7 5C-TerB cells transfected with VC or Tus expression plasmids using the indicated primer pairs. Data shows the fold enrichment relative to IgG controls (n=3).

γH2AX phosphorylation at Tus-TerB is ATR-dependent

(A) MCF7 5C-TerB cells transfected with VC or HA-Tus were treated for 4hrs with 2mM HU before lysis and fractionation (whole cell extract (WCE), soluble fraction and chromatin fraction). Total and phosphorylated protein levels were examined by immunoblotting as indicated. Tus expression was confirmed in WCE. (B) Depletion of pATR Th1989 in MCF7 5C-TerB cells treated with 2mM HU +/- 40 nM of ATR inhibitor was examined with immunoblotting. (C) Schematic of MCF7 5C-TerB depicting the positions of the primer pairs with respect to the integrated TerB locus. (D-E) γH2AX levels were analyzed by ChIP-qPCR in MCF7 5C-TerB cells transfected with (D) VC expression plasmid (E) Tus expression plasmid, +/- 4hrs treatment with ATRi with indicated primer pairs. Data shows the fold enrichment over the IgG controls (n=3).

A local ATR-dependent checkpoint is activated by the Tus-TerB RFB

A model depicting the cellular response to a site-specific DSB using CRISPR-Cas9 and a global replication stress with hydroxyurea, both of which lead to a DNA damage response (DDR, yellow triangles), increased gH2AX (green) levels globally in the cell and, if left unresolved, can alter cell cycle progression. In contrast, the site-specific replication fork block, Tus/TerB, elicits a local ATR dependent DNA damage response which is responsible for the phosphorylation and accumulation of γH2AX at the stalled site. This local signaling does not affect the progression of the cell cycle is not altered during the local checkpoint response.

Generation of the MCF7 5C-TerB cell line

Schematic of the integration of pWB15 in MCF7 cells to generate MCF7 5C-TerB. pWB15 contains two TerB cassettes (grey arrows). Each TerB cassette contains 5 tandem TerB sequences, which are in the non-permissive orientation in pWB15 (grey arrow facing away from each other. The plasmid was linearized by digesting with PvuI for integration into MCF7 cells. The single copy integrant was confirmed using whole genome sequencing and found to be integrated at Chromosome 12.

Expression of Tus in MCF7 5C-TerB cells

(A) Schematic of Doxycycline (Dox) inducible expression of Myc-NLS-TUS-SNAP. (rtTA = reverse tetracycline trans activator; TRE = tetracycline response element). (B) Immunoblot of MCF7 5C-TerB cells stably expressing Dox inducible Myc-NLS-TUS-SNAP. (C-D) Replication profiles shown as the percentage of molecules with IdU incorporation at each 5 Kb interval in the 200 Kb SfiI segment containing TerB sequence, quantified from molecules MCF7 5C-TerB in Figure 2C (Tus not expressed) and Figure 2D (Tus expressed).

Enrichment of FANCM at the Ter sequence in the presence of the Tus protein

ChIP using FANCM antibody was performed MCF7 5C-TerB cells transfected with VC or HA-Tus-His plasmids. ChIP-qPCR were conducted using the indicated primer pairs. Data shows the fold enrichment relative to the IgG controls (n=3).

Distinct patterns of γH2AX enrichment at a Cas9-mediated DSB vs the Tus-TerB fork barrier

(A) Schematic of the linearized TerB plasmid (pWB15) integrated as a unique copy into MCF7 cells (MCF7 5C-TerB) with the Cas9-binding site (green protein). Blue triangles: Cas9 cut site. Black half-arrow heads depict PCR products expected from primer pair (PP10) used in quantitative PCR (qPCR) are shown. (B) Schematic depicting the Cas9 cut site (blue arrows), site-specific PCR primers (black half-arrowhead) and predicted size of cleavage products. (C) PCR analysis by T7 assay of MCF7 5C-TerB cells transfected with Cas9-sgRNA RNP complex. (D) γH2AX levels along the integrated TerB plasmid were analyzed by ChIP-qPCR in MCF7 5C-TerB cells transfected with VC, Tus or Cas9 expression plasmids using the indicated primer pairs. Data shows the fold enrichment relative to IgG controls (n=2).

Genome-wide gH2AX foci were unaffected with Tus expression

(A) Representative images of gH2AX foci across stated conditions. Bars, 10 μm (B) Average number of gH2AX foci per nucleus in conditions stated. Cells treated with or without 2mM HU for 4 hours. (n=3, ≥300 cells per experiment)

Cell cycle progression were unaffected with Tus expression

(A) Cell cycle analysis and (B) quantification was performed using flow cytometry following staining with propidium iodide staining in MCF7 5C-TerB cells under the stated conditions. Cells treated with or without 2mM HU for 4 hours. (n=3, ns: non-significant)