Fig. 1: Direct observation and quantification of DNA G4 structures and their association with replisomes.

a, d Representative epifluorescence (upper left) and SMLM (lower right) images of a single S-phase U2OS nucleus labeled for a nascent DNA (using EdU, red), PCNA (blue), and MCM (green); and d nascent DNA (using EdU, red), G4 (blue), and MCM (green). Scale bar, 2 µm. b, e Schematic illustrations of how TC analysis recognizes triplet patterns from a nucleus. Each red, blue (yellow for G4), and green molecules can form triangles (connected by pale gray lines). If a specific pattern is repeatedly found (dark, bold triangles), its population is distinct from stochastic triplets and therefore is identified as a TC triplet, as shown on right. b shows the TC triplet derived from a, while e shows the TC triplet derived from d. c, f Schematic illustrations of the molecular organizations of “All-Replisome” (without (i) or with (ii) G4 association) resolved by TC analysis of EdU, PCNA, and MCM, as represented in a and b (c), and “G4-Replisome” resolved by TC analysis of EdU, G4, MCM, as represented in d and e (f). g Overlaid TC triplets of EdU, G4, and MCM from multiple non-treated (NT) or 1 h, 200 nM APH-treated cells statistically describe the molecular organization of these three species. Circle size of each TC triplet represent the frequency of G4-Replisomes from a single nucleus. The TC triplets are aligned onto the same EdU-MCM plane to define the positions of G4s relative to the replisome complex. h Frequency of G4-Replisomes in NT or APH-treated cells. Individual data points represent result from single cell. Black horizontal line and box height indicate mean ± SD. Values on graph indicate p-values of unpaired two-sample t-tests between NT and APH-treated cells. i Percent change in frequency of G4-Replisomes in APH-treated compared to NT cells. Values on the graph and black horizontal line represent the percent change, box height indicates the propagated s.e.m. For all experiments, number of cells analyzed and TC triplets identified are listed in Supplementary Table 1.