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. 2014 Mar 13;5(5):1326–1337. doi: 10.18632/oncotarget.1796

Figure 4. The Basal level of FANCD2 Monoubiquitination Concerts with MCM3 Mainly at 477-480AA for the Maintenance of Licensed Replication Origins to Fire at a Normal Rate.

Figure 4

A. 477-480aa of MCM3 is responsible for the interaction between MCM3 and FANCD2. Left panel: Schematic representation of a series of mutated MCM3. Right panel: GFP- MCM3 Δ477-480aa cannot interact with endogenous FANCD2. The seven sets of reverse-IP-WB were performed using CRL-1790 cells transfected with a series of deletion constructs respectively. FANCD2 and GFP- MCM3 477-480AA are not detectable in each other's IP pellets (indicated with red frames). B. MCM3 Δ477-480aa affects the number of licensed/MCMs-assembled origins. A low amount of origin DNA is associated with MCM2 proteins in CRL-1790 cells carrying MCM3 Δ477-480aa as compared to the corresponding empty vector transfected control cells. But ORC remains to interact with a similar number of origins tested. (Please see expression levels of MCM3 Δ477-480aa in CRL set of stable cells as well as ORC ChIP in Supplementary Fig. 4A, 4B, respectively). C. In CRL-1790 cell, GFP-MCM3 Δ477-480aa delays origin firing: enlarged intervals between the fired origins. In stably expressed mutant cells (CRL-1790 Δ477-480aa- Supplementary Fig. 4A) the origin density was significantly low as compared to the control cells (T test, p<0.05). Bars show standard error of the mean (SEM). D. Loss of the basal level of FANCD2 monoubiquitination leads to the enlarged intervals between the fired origins in FA cells. PD20 (FANCD2-/-) cells stably expressing wtFANCD2, K561RFANCD2 or empty vector (Supplementary Fig. 1C1) were used to perform DNA fiber assay. The origin density was significantly low in PD20+empty vector or PD20+K561RFANCD2 cells as compared to PD20+wtFANCD2 cells (T test, p<0.05). Bars show standard error of the mean (SEM). E. A working model for the normally monoubiquitinated FANCD2 to act in DNA replication initiation. In each S-phase of normal cell cycle, monoubiquitinated FANCD2 interacts with replication origins as well as the member(s) of MCM complex, such as MCM3, to provide “a temporal and spatial check” for securing an adequate number of licensed-replication origins to fire at a normal rate. Without the normal basal level of FANCD2 monoubiquitination, cells would not have an enough number of licensed origins to initiate a normal replication, thereby overtime rendering genomic instability, aging and cancer, as shown in FA.