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Published September 27, 2018 | Submitted
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Flexibly-oriented double Cdc45-MCM-GINS intermediates during eukaryotic replicative helicase maturation


The core of the eukaryotic helicase MCM is loaded as an inactive double hexamer (DH). How it is assembled into two active Cdc45-MCM-GINS (CMG) helicases remains elusive. Here, we report that at the onset of S phase, both Cdc45 and GINS are loaded as dimers onto MCM DH, resulting in formation of double CMG (d-CMG). As S phase proceeds, d-CMGs gradually mature into two single CMG-centered replisome progression complexes (RPCs). Mass spectra reveal that RPA and DNA Pol α/primase co-purify exclusively with RPCs, but not with d-CMGs. Consistently, d-CMGs are not able to catalyze either the unwinding or de novo DNA synthesis, while RPCs can do both. Using single-particle electron microscopy, we have obtained 2D class averages of d-CMGs. Compared to MCM DHs, they display heterogeneous, flexibly orientated and partially loosened conformations with changed interfaces. The dumbbell-shaped d-CMGs are mediated by Ctf4, while other types of d-CMGs are independent of Ctf4. These data suggest CMG dimers as bona fide intermediates during MCM maturation, providing an additional quality control for symmetric origin activation and bidirectional replication.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. We thank Dr. Costa for sharing unpublished data and discussion, Drs. Stephen Bell, and Li-Lin Du for reagents; Drs. Ning Gao, Hao Wu, Qun He, Yisui Xia and members of the Lou lab for helpful discussion and comments on the manuscript. This work was supported by the National Natural Science Foundation of China 31630005, 31770084, 31771382 and 31271331; the National Basic Research Program (973 Program) of China (2014CB849801); Chinese Universities Scientific Fund 2015TC039 and 2014JD075; Opening Project of the State Key Laboratory of Microbial Resources; Program for Extramural Scientists of the State Key Laboratory of Agrobiotechnology 2018SKLAB6-5. Author contributions: L.L. and Y.Z. performed most of the experiments except for the single-particle EM in Figures 6 and 7, which was carried out by J.Z. All the mass spectrometry analysis was performed by J-H.W., M-Q.D. and Z.L. The authors declare no competing financial interests.

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