A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- Brandeis University, United States
Abstract
Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions, however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.
Data availability
Source data for the plots in Figs. 3, 4, and 7 are found in the file "Champasa_Band_Analysis.xlsx".Source data for the single-molecule experiments is provided as "intervals" files that can be read and manipulated by the Matlab program imscroll, which is publicly available: https://github.com/gelles-brandeis/CoSMoS_Analysis.The source data are archived as doi:10.5281/zenodo.2556799.All remaining data generated or analyzed during this study are included in the manuscript.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (GM52339)
- Stephen P Bell
Howard Hughes Medical Institute (Investigator)
- Stephen P Bell
G Harold and Leila Y Matthews Foundation
- Jeff Gelles
National Cancer Institute (P30-CA14051)
- Stephen P Bell
National Institute of General Medical Sciences (GM81648)
- Jeff Gelles
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Champasa et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding
eLife 8:e45538.
https://doi.org/10.7554/eLife.45538
