G-quadruplex-stalled eukaryotic replisome structure reveals helical inchworm DNA translocation

Abstract

DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork collisions with preformed G4s in the parental DNA using reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by arresting the CMG helicase. Cryo-electron microscopy structures of stalled yeast and human CMG complexes reveal that the folded G4 is lodged inside the central CMG channel, arresting translocation. The G4 stabilizes the CMG at distinct translocation intermediates, suggesting an unprecedented helical inchworm mechanism for DNA translocation. These findings illuminate the eukaryotic replication fork mechanism under normal and perturbed conditions.

Figure 1:. A single G4 in the leading strand template is sufficient to block replication fork progression.

(A) Denaturing (bottom) and native (top) agarose gel analysis of replication products obtained in the reconstituted yeast system on DNA templates harboring no G4 (lanes 1+2), a G4 on the leading strand template (lanes 3+4) or a G4 on the lagging strand template (lanes 5+6). RI: Replication intermediates. (B) Schematic of replication products obtained in (A). Red: Leading strand products; Blue: Lagging strand products.

Figure 2:. The stalled CMG-FPC protects DNA upstream and downstream of the G4.

(A) 3’ exonuclease protection analysis to determine position of the C-tier of yeast CMG-FPC stalled at G4 on the leading strand template. (B) 5’ exonuclease protection analysis to determine position of the N-tier of yeast CMG-FPC stalled at G4 on the leading strand template. (C) Schematic summarizing data from (A) and (B). Green: CMG. Yellow: Tof1-Csm3 (TC). N: N-terminal. C: C-terminal.

Figure 3:. The G4 is lodged inside the central channel of stalled yeast CMG-FPC.

(A,B) Cryo-EM density map of yeast CMG-FPC stalled at G4, state 1 (A) and state 2 (B) colored by subcomplex with Mcm2–7 in green, Cdc45-GINS in blue, Tof1-Csm3 in yellow and DNA in grey. (C,D) Structure of the G4 bound in the central chamber of the MCM ring in state 1 (C) and state 2 (D). Densities corresponding to the DNA are shown as grey isosurfaces and contoured at 2.5 σ. (E,F) Surface representation of MCM central chamber in state 1 (E) and state 2 (F) with Mcm4 and Mcm7 removed for clarity.

Figure 4:. The MCM PS1/H2I loops sterically block passage of the leading strand template at the G4.

(A,B) Surface representation of the constriction from the central cavity to the MCM C-tier for state 1 (A) and state 2 (B) with DNA shown as cartoon, viewed from the N-tier. (C) Interactions between MCM PS1/H2I loops and the G4 in state 1 (C) and state 2 (D), viewed from the side. (E-H) MCM PS1/H2I loops in G4 stall states 1 (E) and 2 (F) and normal fork conformations 1 (G) and 2 (H). Mcm2 is shown in blue, Mcm6, −4, −3, and −7 are shown in green and Mcm5 is shown in orange.

Figure 5:. Nucleotide-bound state of G4-stalled yeast CMG-FPC.

(A,B) MCM C-tier of state 1 (A) and state 2 (B). Bound ATP molecules are shown as orange spheres, bound ADP molecules are shown as magenta spheres and ssDNAs are shown as grey surfaces. (C,D) MCM C-tier ATPase sites in state 1 (C) and state 2 (D).

Figure 6:. The G4 stall mechanism is conserved in the human replisome.

(A) Denaturing (bottom) and native (top) agarose gel analysis of replication products obtained in the reconstituted human system on DNA templates harboring a G4 on the leading strand template (lanes 1–3) or a G4 on the lagging strand template (lanes 4–6). A random B-form DNA fragment was ligated into the template in lanes 7–9, while no fragment was inserted into the template in lanes 10–12. * indicates position of leading strand products resulting from replisome run-off at template nicks, which coincide with stalled products. (B) Schematic of replication products obtained in (A). (C) Cryo-EM density map of human CMG-FPC stalled at G4 colored by subcomplex. (D) Structure of the G4 bound in the central chamber of the MCM ring. Density corresponding to the DNA is shown as a grey isosurface and contoured at 2.5 σ. (E) Interactions between MCM PS1/H2I loops and bound G4, viewed from the side. (F) Surface representation of MCM central chamber with MCM4 and MCM7 removed for clarity (G) Surface representation of the access to the MCM C-tier with DNA shown as cartoon, viewed from the N-tier. (H) MCM C-tier with bound ATP molecules shown as orange spheres, bound ADP molecules shown as magenta spheres and ssDNAs shown as a grey surface.