Figure 1. ATR prevents RPA accumulation at replication forks during unperturbed replication.

(A and B) Representative multi-color SMLM images of replication factors PCNA, RPA, and MCM in S phase cells that were either non-treated (A, NT) or treated with ATR inhibitor (B, ATRi). Magnified panels for each image show representative triplet patterns of individual replisomes.

(C and D) Schematic illustration of the data mining workflow using TCF based approach for recognition of triplet patterns. Any molecule i from the Red, j from the Green, and k from the Blue channel can form a triplet $\text{Δ}ijk$, defined by their distances $\left[d_{\text{RG}},d_{\text{RB}},d_{\text{BG}}\right]$. The occurrence of all these triplets (e.g. $\text{Δ}ijk,\text{Δ}ijm,\text{Δ}ljq$, …) is then counted as a function of their triple-wise distances (i). When many molecules of the three species form a similar triplet pattern (i.e. many triplets with similar $\left[d_{\text{RG}},d_{\text{RB}},d_{\text{BG}}\right]$), the probability density distribution of such triplet rises at $\left[d_{\text{RG}},d_{\text{RB}},d_{\text{BG}}\right]$ from the level of uncorrelated random patterns (ii) (Methods S1).

(E) The most populated triplet pattern as derived from the single-cell TCF distribution (D, ii).

(F and G) Workflow for pooled representation of the TCF-resolved single-replisome configurations; The single-replisome configuration is resolved for each cell (F) and the configurations for multiple cells are overlaid (G).

(H) Quantification of the levels of RPA_fork for the TCF-resolved single replisome configurations shown in (G) reveals an ATRi-induced RPA accumulation at forks. Mean values are shown as the black bar, N = 193 and 177 for NT and ATRi, respectively.

(I and J) Kinetics of ATRi-mediated RPA accumulation at forks. Mean values and the 1^st and 3^rd quartile are marked as black and colored bars, respectively. N = 193, 53, 70, and 177 for NT, 60, 120, and 180 minutes treatment in (I), respectively; N = 193, 177, 72, 68, and 79 for NT, 0, 60, 90, 120 minutes release in (J), respectively.