Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published August 2020 | Supplemental Material
Journal Article Open

Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks


The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.

Additional Information

© 2019 Springer Nature Switzerland AG. Received: 1 April 2019; Revised: 17 September 2019; Accepted: 7 October 2019; First Online 15 November 2019. We thank Dr. Costa for sharing unpublished data and superposed CMG-Ctf4 structure shown in Fig. 6e; Dr. Karim Labib for insightful discussion; Drs. Karim Labib, Stephen Bell, and Li-Lin Du for reagents; Drs. Ning Gao, Hao Wu, Qun He, Yisui Xia, Ms. Yawen Bai, and members of the Lou lab for help and comments on the manuscript. This work was supported by the National Natural Science Foundation of China (http://www.nsfc.gov.cn/) 31630005 and 31770084 to HL, 31771382 to QC, 31800066 to JZ; the China Postdoctoral Science Foundation (http://jj.chinapostdoctor.org.cn/) 2018M640201 to JZ; the National Basic Research Program (973 Program) of China (http://www.most.gov.cn/) 2014CB849801 to MQD; Program for Extramural Scientists of the State Key Laboratory of Agrobiotechnology (http://en.cau.edu.cn/colleges/biological-science/) (2018SKLAB6-5) to MQD; Opening Project of the State Key Laboratory of Microbial Resources (http://english.im.cas.cn/) to HL. The authors declare no competing financial interests.

Attached Files

Supplemental Material - 18_2019_3333_MOESM1_ESM.docx


Files (1.5 MB)
Name Size Download all
1.5 MB Download

Additional details

August 22, 2023
December 22, 2023