Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling

Abstract

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.

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.

Fig. 2. Effects of G4 formation on replisome structure and progression.

a, e Overlaid TC triplets of RPA, PCNA, MCM (a) or EdU, PCNA, MCM (e) (top, All Replisomes), and RPA, G4, MCM (a) or EdU, G4, MCM (e) (bottom, G4-Replisomes) from multiple NT or APH-treated cells. Circle size of each TC triplet represents the local density of RPA (a) or EdU (e) from a given nucleus. For a, the TC triplets are aligned onto the same EdU-MCM (top) or G4-MCM (bottom) plane to define the positions of RPA relative to the replisome complex. For e, the TC triplets are aligned using MCM as the center to better visualize the relative magnitude of EdU. b, f Comparison of the local densities of RPA (b) or EdU (f) within All-Replisomes (light gray) or G4-Replisomes (dark gray) in NT and APH-treated cells. Error bars indicate mean ± s.e.m. Values on graph indicate p-values of unpaired two-sample t-tests between All-Replisomes and G4-Replisomes. Corresponding data plots showing the data distributions are presented in Supplementary Fig. 1d, e, h, i. c, g Percent change in the densities of RPA (c) or EdU (g) at All-Replisomes or G4-Replisomes in APH-treated compared to NT cells. Values on the graph and black horizontal line represent the respective percent changes, box height indicates the propagated s.e.m. d, h Schematic illustrations showing that the spontaneously folded G4 structure at an active replication fork blocks RPA recruitment onto ssDNA during regular replication (d) and locally hinders DNA synthesis (h). For all experiments, number of cells analyzed and TC triplets identified are listed in Supplementary Table 1.

Fig. 3. FANCJ suppresses accumulation of G4 structures within replisomes.

a, d, g Overlaid TC triplets of EdU, G4, MCM (a), EdU, PCNA, MCM (d), or EdU, G4, MCM (g), from multiple NT or 1 h, 20 μM PDS-treated S-phase U2OS cells transfected with control (siCTRL) or FANCJ (siFANCJ) siRNA. Circle sizes of each TC triplet represents the local density of G4-Replisomes (a), EdU at All-Replisomes (d), or EdU at G4-Replisomes (g) from a given nucleus. For a, the TC triplets are aligned onto the same EdU-MCM plane to define the positions of G4 relative to the replisome complex; for d, g, the TC triples are aligned using MCM as the center to better visualize the relative magnitude of EdU. b, e, h Frequencies of G4-Replisomes (b), EdU at All-Replisomes (e), or EdU at G4-Replisomes (h) in NT or PDS-treated siCTRL or siFANCJ cells. Individual data points represent result from a single nucleus. Black horizontal line and box height indicate mean ± SD. Values on graph indicate p-values of unpaired two-sample t-tests between NT and PDS-treated cells. c, f, i Percent change in the densities of G4-Replisomes (c), EdU at All-Replisomes (f), or EdU at G4-Replisomes (i) in siCTRL or siFANCJ PDS-treated compared to NT cells. Values on the graph and black horizontal line represent the respective percent changes, 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.

Fig. 4. Deregulated G4-Replisomes resist RPA loading.

a, d Overlaid TC triplets of RPA, PCNA, MCM (a) or RPA, G4, MCM (d) from multiple NT or 4 h, 20 μM PDS-treated siCTRL or siFANCJ cells. Circle size of each TC triplet represents the local density of RPA from a given nucleus. The TC triplets are aligned onto the same PCNA-MCM (a) or G4-MCM (d) plane to define the positions of RPA relative to the replisome complex. b, e Local densities of RPA at All-Replisomes (b) or G4-Replisomes (e) in NT or PDS-treated siCTRL or siFANCJ cells. Individual data points represent result from a single nucleus. Black horizontal line and box height indicate mean ± SD. Values on graph indicate p-values of unpaired two-sample t-tests between NT and PDS-treated cells. c, f Percent change in the densities of RPA at All-Replisomes (c) or G4-Replisomes (f) in siCTRL or siFANCJ PDS-treated compared to NT cells. Values on the graph and black horizontal line represent the respective percent changes, 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.

Fig. 5. Interplay between FANCJ and RPA on G4-resolution.

a Illustration of the FANCJ/RPA-mediated destabilization of a single G4 substrate monitored by smFRET. b Representative single-molecule trajectories of the L1 G4 substrates in the presence or absence of 10 nM RPA and/or 100 pM FANCJ+ATP. c FRET histograms of L1 G4 in the presence of indicated concentrations of RPA with either no (left panel) or 100 pM FANCJ (right panel). d Fractions of unfolded L1 G4 monitored by smFRET as a function of RPA concentration with (red) or without (black) the addition of FANCJ. Error bars represent mean ± s.e.m. e Unfolding (top) and folding (bottom) rates for L1 G4 as a function of RPA concentration with (red) or without (black) the addition of FANCJ. Error bar represents the SD of exponential fit. f Fraction of unfolded L1 G4 in indicated conditions monitored by smFRET. Data collected from two experiments, shown as black datapoints, were pooled together for analysis. Error bars represent mean ± SD. For all smFRET experiments, a minimum of 100 smFRET trajectories from two independent experiments were used for analysis.

Fig. 6. Unresolved G4-Replisome accumulation causes DNA damage.

a Overlaid TC triplets of γH2AX, G4, MCM from multiple NT or 4 h, 20 μM PDS-treated siCTRL or siFANCJ cells. Circle size of each TC triplet represents the local density of γH2AX from a given nucleus. The TC triplets are aligned onto the same G4-MCM plane to define the positions of γH2AX relative to the replisome complex. b Local densities of γH2AX at G4-Replisomes in NT or PDS-treated siCTRL or siFANCJ cells. Individual data points represent result from a single nucleus. Black horizontal line and box height indicate mean ± SD. Values on graph indicate p-values of unpaired two-sample t-tests between NT and PDS-treated cells. c Percent change in the densities of γH2AX at G4-Replisomes in siCTRL or siFANCJ PDS-treated compared to NT cells. Values on the graph and black horizontal line represent the respective percent changes, 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. d Model of FANCJ/RPA-mediated G4 regulation and consequences at replicating forks: during DNA replication at G4 motifs, the exposed ssDNA stretches may fold into G4 structures. RPA can destabilize some G4s, allowing for seamless continuation of replication fork progression. For G4s with higher thermostability (RPA-Resistant G4s), RPA alone would not be sufficient to destabilize them, therefore requiring facilitation by FANCJ, whose initial unwinding of G4 facilitates the loading of RPA onto ssDNA, thereby activating a proper replication stress response to promote replication fork restart. Accordingly, loss of FANCJ results in accumulation of RPA-resistant G4s at forks, leading to persistent fork uncoupling and stalling, the silencing of replication stress signaling, eventually leading to DNA damage accumulation at G4-Replisomes.