Quantitative Mcm Perturbation in Mice Figure 6. Inhibition of Chaos3 cancers by MCM3 reduction. (A) Kaplan-Meier graphs of cohorts of the indicated genotypes. C3 = Mcm4Chaos3; M# = Mcm#Gt. In the left panel, the experiment was terminated at 12 months, with ,1/3 animals tumor-free and healthy at the time (see ‘‘B’’). Unless otherwise indicated, the cohorts contained both sexes. (B) Pie charts of cancer types in mice from ‘‘A.’’ COD = cause of death; Unkn = unknown. Classification of cancer types was assigned during necropsy, not from histological analysis. doi:10.1371/journal.pgen.1001110.g006 samples by Western analysis (not shown). Therefore, we quantified MCM2 during the cell cycle by flow cytometric analysis of nuclei from 7 replicate MEF cultures. Similar to WT MEFs (examples in Figure 1B), NIH3T3 cells showed a decrease of nuclear MCM2 during S phase progression (Figure 7D, left panel). However, all genotypes with in the Mcm4Chaos3/Chaos3 background had a reduced decline. Thus, for comparative quantitation across genotypes, we compared the levels of MCM2 levels at the beginning of G1 vs. that in S phase (regions used for these calculations are indicated in the left panel), using the calculation described in the Figure 7 legend. The data are graphed in the right panel. The data revealed that regardless of genotype, the difference in average amounts of nuclear MCM2 at the beginning and end of G1 (DG1) did not vary. Compared to Mcm4Chaos3/Chaos3, cells lacking 1 dose of Mcm2 had relatively lower levels of S phase MCM2 (DS) compared to early G1. Additional removal of an Mcm3 dose partially rescued the DS value, indicating that these cells had ,16% more nuclear MCM2 in S phase compared to Mcm4Chaos3/Chaos3 cells hemizygous for Mcm2 alone, despite overall reduced MCM2 levels in the cell (Figure 7B, left panel). Discussion MCM2-7 proteins exist abundantly in proliferating cells and are bound to chromatin in amounts exceeding that required to license all replication origins that initiate DNA synthesis [9–12,14]. The role of excess chromatin-bound MCM2-7 has been a mystery referred to as the ‘‘MCM paradox’’ [27], perpetuated by observations that drastic MCM reductions in certain systems can be compatible with normal DNA replication or cell proliferation [13,28–30]. However, these circumstances are not universal, and reductions are not entirely without consequences. Early studies showed that a reduction in MCMs resulted in decreased usage of certain ARSs [12] and conferred genome instability [31] in yeasts. In cell culture systems, depletion of certain MCMs have been found to cause cell cycle defects, checkpoint abberations and GIN [13,16–17,29,32]. Recent work has shed light on aspects of the MCM paradox. Using Xenopus egg extracts attenuated for licensing by addition of geminin (an inhibitor of CDT1, which is required for MCM loading onto origins), one study proposed that excess chromatin- bound MCM2-7 complexes license ‘‘dormant’’ origins that can be activated to rescue stalled or damaged replication forks, a situation that can become important under conditions of replication stress [11]. Similar results were subsequently reported for human cells depleted of MCMs by siRNA [15–16], and for replication stressed MCM2-deficient MEFs [21]. Our finding that nuclear MCM2 levels decrease as S-phase progresses, and moreso in WT than in Mcm4Chaos3/Chaos3 MEFs, is consistent with the dormant origin hypothesis. The decrease may reflect displacement of dormant hexamers by active replisomes, followed by subsequent degrada- PLoS Genetics | www.plosgenetics.org 10 September 2010 | Volume 6 | Issue 9 | e1001110