Cell-Cycle-Regulated Interaction between Mcm10 and Double Hexameric Mcm2-7 Is Required for Helicase Splitting and Activation during S Phase
Mcm2-7 helicase is loaded onto double-stranded origin DNA as an inactive double hexamer (DH) in G1 phase. The mechanisms of Mcm2-7 remodeling that trigger helicase activation in S phase remain unknown. Here, we develop an approach to detect and purify the endogenous DHs directly. Through cellular fractionation, we provide in vivo evidence that DHs are assembled on chromatin in G1 phase and separated during S phase. Interestingly, Mcm10, a robust MCM interactor, co-purifies exclusively with the DHs in the context of chromatin. Deletion of the main interaction domain, Mcm10 C terminus, causes growth and S phase defects, which can be suppressed through Mcm10-MCM fusions. By monitoring the dynamics of MCM DHs, we show a significant delay in DH dissolution during S phase in the Mcm10-MCM interaction-deficient mutants. Therefore, we propose an essential role for Mcm10 in Mcm2-7 remodeling through formation of a cell-cycle-regulated supercomplex with DHs.
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Received: March 11, 2015; Revised: September 22, 2015; Accepted: November 3, 2015; Published: December 10, 2015. We thank Drs. Bruce Stillman, Karim Labib, Li-Lin Du, Quanwen Jin, Wei Xiao, and Junbiao Dai for reagents and Drs. John Diffley, Karim Labib, Philippe Pasero, Qun He, Li-Lin Du, Jing Li, and members of the H.L. lab for helpful discussion and comments on the manuscript. This work was supported by National Natural Science Foundation of China 31271331 and 31071095 to H.L. and 31200052 to Q.C., Program for New Century Excellent Talents in University of China NCET-09-0733 to H.L., NIH grant GM080338 to X.S.C., Research Fund for the Doctoral Program of Higher Education of China 20120008110017, Project (2014SKLAB1-7) and Program for Extramural Scientists (2015SKLAB6-26) of the State Key Laboratory of Agrobiotechnology, and Chinese Universities Scientific Fund 2015TC039 and 2014JD075. Author Contributions: H.L., Y.Q., Y.X., and J.L.C. conceived and designed research. Y.X. and Y.Q. purified the native MCM complexes. Y.Q., Y.X., L.L., J.C., and Q.C. performed other experiments. Y.Q. and Z.L. did mass spectrometry analysis. H.L., J.L.C., X.S.C., Y.Q., and Y.X. analyzed the data and wrote the paper.
Published - PIIS2211124715013182.pdf
Supplemental Material - mmc1.pdf
Accepted Version - nihms878338.pdf