#Rootsmagic 7 dna entry full#

#Rootsmagic 7 dna entry full#

The pre-RC-induced ATPase activity is shown in red (ATPase activity of the full pre-RC reaction with the ATPase activity of the individual components subtracted).Ī rapamycin-induced linkage between Mcm2 and Mcm5 affects pre-RC establishment but not maintenance. Together, ORC, Cdc6, Cdt1, and MCM2–7 induced a strong ATPase activity. The ATP hydrolysis rates were determined for the indicated proteins in the presence of ARS1 origin DNA. ( F) Analysis of ATPase activities during pre-RC assembly with wild-type (wt) MCM2–7 or MCM2–7–M2/M5 in the presence or absence of rapamycin. ( E) Pre-RC reactions using an alternative MCM2–7–M2/M5 construct were assembled in the presence or absence of rapamycin and ATP, washed with low salt (L) or high salt (H), and analyzed by silver staining. Asterisks mark nonspecific IgG-related bands.

A 30% input is shown along with 100% of the immunoprecipitate. ( D) The MCM2–7–Cdt1 interaction analysis used IgG control beads or MCM2–7 (HA-Mcm3) coupled to anti-HA beads. ( C) Illustration showing the rapamycin-induced linkage between neighboring Mcm subunits.

( B) Pre-RC reactions using MCM2–7–M2/M5 were assembled in the presence or absence of rapamycin and ATP, washed with low salt (L) or high salt (H), and analyzed by silver staining. The smallest subunit of the Orc1–6 complex stains only weakly by silver staining. ( A) Pre-RC reactions using MCM2–7 and five MCM2–7–FKBP/FRB protein complexes were assembled in the presence or absence of rapamycin and ATP, washed with low-salt (L) or high-salt (H) buffer, and analyzed by silver staining. Only MCM2–7–M2/M5 in the presence of rapamycin displays a helicase loading defect.

Published by Cold Spring Harbor Laboratory Press. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.ĭNA licensing DNA replication cancer genomic stability pre-RC replicative helicase. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability.