A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding.

ELife
Kanokwan ChampasaStephen P Bell

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.

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Citations

May 6, 2020·Biochemical Society Transactions·Jacob S Lewis, Alessandro Costa
Jul 10, 2021·Frontiers in Molecular Biosciences·Jian ZhouGen Wu
May 17, 2021·DNA Repair·Yo-Chuen Lin, Supriya G Prasanth
Oct 27, 2021·Nature·Michael Jenkyn-BedfordTom D Deegan

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Methods Mentioned

BETA
FRET
gel filtration
gel-filtration
size-exclusion chromatography
E
crosslinking studies

Software Mentioned

PSIPRED
Quick2D
Matlab ( MATHWORKS )
ImageJ
IUPRED
ebfret

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