Treffer: G-quadruplex–stalled eukaryotic replisome structure reveals helical inchworm DNA translocation.

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. Editor's summary: Naturally occurring obstacles in chromosomal DNA can challenge the faithful propagation of the genome across generations. Batra et al. determined that noncanonical DNA secondary structures called G-quadruplexes can impede DNA copying by arresting the replicative DNA helicase known as CMG (Cdc45-MCM-GINS), which serves as the central engine of the molecular machinery that copies chromosomal DNA. The CMG helicase moves along DNA through a previously unrecognized inchworm-like mechanism. These observations uncover fundamental principles of the chromosome replication process that are essential for the maintenance of genome stability. —Di Jiang INTRODUCTION: Cellular genomes are replicated by large, multisubunit complexes, called replisomes, which orchestrate the unwinding of the parental DNA and synthesis of the daughter strands at replication forks. Cellular replisomes in all domains of life are assembled around ring-shaped hexameric DNA helicases that topologically encircle DNA to drive the processive unwinding of the parental DNA. The eukaryotic replicative DNA helicase, CMG (Cdc45-MCM-GINS), is composed of the ring-shaped heterohexameric MCM ATPase and the noncatalytic cofactors, Cdc45 and GINS. At replication forks, CMG encircles and translocates along the leading strand template using DNA-binding hairpin loops that line its central channel, while sterically excluding the lagging strand template from entering the channel. Notably, CMG-driven DNA unwinding is frequently challenged by physical obstacles in the chromosomal template, including non–B-form DNA secondary structures, such as G-quadruplexes (G4s), which can cause replication stress and pose a threat to genome stability. RATIONALE: Our previous studies established that G4s can impede replisome progression by inhibiting the CMG helicase. However, the molecular mechanism of DNA translocation by CMG and how G4s might interfere with CMG activity is not understood. Elucidation of these processes is critical to understanding both normal replisome function and the causes of replication stress. To this end, we took advantage of our ability to reconstitute yeast and human replisomes with purified proteins, which allows for a detailed biochemical and structural characterization of replisomes stalled at G4s in the parental DNA template. RESULTS: We demonstrate that a single G4 can arrest DNA replication by stalling the translocation cycle of the CMG helicase. Our cryo–electron microscopy (cryo-EM) structures of yeast and human CMG stalled at a G4 in the leading strand template reveal that the G4 is fully folded and lodged within the central channel of CMG. Thus, the G4 is effectively sequestered away from G4-resolving helicases inside the stalled replisome, raising questions on potential fork restart mechanisms. We resolved two distinct conformations of G4-stalled CMG. The first closely resembles previously reported structures of CMG bound to normal forked DNA, recapitulating the protein-DNA interactions inside the CMG channel necessary for both DNA translocation and unwinding. Our second structure, by contrast, differs from previously determined CMG conformations and is consistent with an intermediate state along the translocation pathway. Our analysis provides evidence that CMG utilizes a previously uncharacterized mechanism to propel the 3′-to-5′ movement of the replication fork. This mechanism features oscillatory spiral-to-planar transitions of DNA-binding loops inside the CMG central channel, contrasting with the sequential rotary mechanism proposed previously for the homohexameric replicative DNA helicases in bacterial systems. CONCLUSION: Our work provides definitive physical evidence for how DNA secondary structures can pose formidable barriers to the chromosomal DNA replication machinery. Additionally, our structures of CMG stalled at G4-containing replication forks unexpectedly point to a DNA translocation mechanism that is distinct among hexameric helicases. Further studies will be important for understanding the kinetics of this process and how G4-induced fork arrest may be resolved to allow for the completion of chromosome replication. G4s stall the eukaryotic replicative helicase.: The eukaryotic replisome is assembled around the replicative DNA helicase, CMG. A single G4 can arrest DNA replication by stalling the CMG translocation cycle. Cryo-EM structures of G4-stalled fork protection complex (FPC)–bound CMG reveal a translocation cycle intermediate with a distinct conformation of the MCM presensor 1 and helix 2 insert DNA-binding hairpin loops. [ABSTRACT FROM AUTHOR]

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