Figure 4

Efficient depletion of Mcm4-aid protein in the MCM complex on chromatin causes dissociation of Mcm6 from chromatin and replication fork arrest. (A) Experimental scheme. HM3086 cdc25-22 mcm4-aid P _adh15 -skp1-AtTIR1-2NLS cells were arrested at the G2/M boundary by incubation at 36°C (indicated as G2) and released at 25°C in the presence of hydroxyurea (HU) (12 mM) to arrest them at early S-phase. After 2 h of incubation with HU (HU2h), the cells were incubated with or without auxin (0.5 mM) for 1 h (HU3h+A, HU3h-A). After removal of HU by filtration, the cells were cultured with or without auxin (0.5 mM) and their DNA contents were analyzed. (B) Immunoblotting of MCM components. The amounts of proteins in the extracts prepared at G2, HU2h, and HU3h with or without auxin were analyzed by immunoblotting with antibodies against Mcm4, Mcm2, Mcm3, Mcm5, Mcm6, Mcm7 and TAT1. Position of phosphorylated Mcm4 in the presence of HU is indicated by an asterisk (*). (C) Dissociation of Mcm6 from the replication origin by depletion of Mcm4-aid. Positions of ars2004 and non-ARS1 are indicated above the panel with the distance from the left end of chromosome II. ChIP assay with anti-Mcm6 was carried out at G2, HU2h, HU3h-A (-auxin) and HU3h+A (+auxin). Recoveries of ars2004 and non-ARS1 fragments measured by real-time PCR are presented. The error bar shows the standard deviations for three independent ChIP analyses. (D) DNA contents of cells released from HU block upon depletion of Mcm4. Cells released from HU block were incubated in the presence or absence of auxin (0.5 mM), and the DNA contents were analyzed. Positions of 1C and 2C DNA contents are shown.