A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells

Joint Public Review:

The authors report the first use of the bacterial Tus-Ter replication block system in human cells. A single plasmid containing two divergently oriented five-fold TerB repeats was integrated on chromosome 12 of MCF7 cells. ChIP and PLA experiments convincingly demonstrate the occupancy of Tus at the Ter sites in cells. Using an elegant Single Molecule Analysis of Replicated DNA (SMARD) assay, convincing data demonstrate the replication block at Ter sites dependent on the presence of the protein. As an orthogonal method to demonstrate fork stalling, ChIP data show the accumulation of the replicative helicase component MCM3 and the repair protein FANCM around the Ter sites. It is unclear whether the Ter sites integrated by a single copy plasmid have any effect on the replication of this region but the data show that the observed effects are dependent on expression of the Tus protein. The SMARD data do not reveal what proportion of forks are arrested at Tus/Ter, or how long the fork delay is imposed. Fork stalling led to a highly localized gammaH2AX response, as monitored by ChIP using primer pairs spread along the integrated plasmid carrying the Ter sites. This response was shown to be dependent on ATR using the ATR inhibitor VE-822. This contrasts with a single Cas9-induced DSB between the two Ter sites, which causes a more spread gammaH2AX response. While this was monitored only at a single distal site, the difference between the DSB and the Tus-induced stall is very significant. Interestingly, despite evidence for ATR activation through the gammaH2AX response, no evidence for phosphorylation of ATR-T1989, CHK1-S345, or RPA2-S33 could be found under fork stalling conditions. The global replication inhibitor hydroxyurea (HU) elicited phosphorylation of ATR-T1989, CHK1-S345, or RPA2-S33. In this context, it would have been of interest to examine if a single DSB in the Ter region leads to phosphorylation of ATR-T1989, CHK1-S345, or RPA2-S33 and cell cycle arrest. It is not shown whether the replication inhibitor HU leads to the same widely spread gamma H2AX response. Overall, this is a well written manuscript, and the data provide convincing evidence that the Tus-Ter system poses a site-specific replication fork block in MCF7 cells leading to a localized ATR-dependent DNA damage checkpoint response that is distinct from the more global response to HU or DSBs.

Author Response

“It is unclear whether the Ter sites integrated by a single copy plasmid have any effect on the replication of this region but the data show that the observed effects are dependent on expression of the Tus protein.”

-The lack of perturbation of the TerB sequence on fork progression has extensively been studied previously in both Willis et al, 2014 and Larsen et. al, 2014. Furthermore, as the detection of the SMARD signal at the TerB sites is dependent on the 7.5kb probe that spans the TerB sites (orange probe, Fig 2B & 2D), it would be impossible to study the effect on replication in this region, with and without the integration of the single copy plasmid.

“The SMARD data do not reveal what proportion of forks are arrested at Tus/Ter, or how long the fork delay is imposed.”

-The percentage of fork stalling at the TerB sites, with and without Tus expression, has been quantified in Figure 2E & 2F. Essentially, 36% forks stall at the TerB block, i.e. 18% of the forks stall in both the 5’ to 3’ (orange) and 3’ to 5’ (blue) direction when the Tus-TerB block is active.

“It is not shown whether the replication inhibitor HU leads to the same widely spread gamma H2AX response.”

-While we have not shown gH2AX accumulation via ChIP after HU treatment, Supplementary Figure 5A & 5B clearly show increased gH2AX foci when the cells are treated with HU, suggesting a global replication stress response that is in stark contrast to the response to Tus-TerB.