A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding
Name
elife-45538-v2.pdf
Description
Published version
Size
2.68 MB
Format
Adobe PDF
Checksum (MD5)
62f267a774f59f52875578c4c3dc86d2
Author(s)
Champasa, Kanokwan
Blank, Caitlin
Friedman, Larry J
Gelles, Jeff
Bell, Stephen P
Date Issued
August 2019
Journal
eLife
Publisher
eLife Sciences Publications, Ltd
Citation
Champasa, Kanokwan et al. "A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding." eLife 8: e45538 © 2019 Champasa et al.
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution 4.0 International license
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.7554/elife.45538
