Author Summary Proper replication of the genome is essential for mainte- nance of the genetic material and normal cell proliferation. DNA replication can be compromised by exogenous factors and genetic disruptions. Such compromise can lead to disease such as cancer, which is characterized by genomic instability (an elevated mutation rate). Because the DNA replication apparatus is essential, relatively little is known about how genetic variants impact the health of whole animals. In this report, we studied mice bearing combinatorial mutations in a component of the replication apparatus, the MCM2-7 helicase. MCM2-7 is a complex of 6 proteins that are essential for initiating DNA replication along chromosomes, and to unwind the DNA during DNA replication. We find that although cells have excess amounts of MCM2-7 to support proliferation under normal circumstances, that incremental MCM depletions have multiple drastic effects upon the whole animal, including embryonic lethality, stem cells defects, and severe cancer susceptibility. Additionally, we report that mouse cells regulate and coordinate the levels of usable MCM proteins, both at the level of synthesis and also by regulating access to chromatin. The implication is that genetic variants that impact MCM levels, even to a minor degree, can translate into disease. that relatively modest decreases in any of the MCMs may be sufficient to cause cancer susceptibility, developmental defects, and GIN [20]. Here, we report that genetically-induced reductions of MCM levels in mice, achieved by breeding combinations of MCM2-7 alleles, caused several health-related defects including increased embryonic lethality, GIN, cancer susceptibility, growth retarda- tion, defective cell proliferation, and hematopoiesis defects. Remarkably, genetic reduction of MCM3, which mediates nuclear export of excess MCM2-7 complexes in yeast [22], rescued many of these defects, presumably attributable to observed increases in chromatin-bound MCM levels. These data suggest that relatively minor misregulation or destabilization of MCM homeostasis can have serious consequences for health, viability and cancer susceptibility of animals. Results Mcm4Chaos3/Chaos3 cells exhibit pan-reduction of total and chromatin-bound MCM2-7 due to decreased mRNA levels To extend previous findings that Mcm4Chaos3Chaos3 cells exhibited decreases in MCM4 and MCM7 protein, and to determine if the decreased levels were differentially compartmentalized in the cell, we quantified soluble and chromatin-bound MCM2-7 levels in mouse embryonic fibroblasts (MEFs) by Western blot analysis. As shown in Figure 1A, all MCMs were decreased in both compartments by at least 40% compared to WT cells. Because Mcm4Chaos3/Chaos3 MEF cultures have slightly decreased prolifera- tion and G2/M delay (Figure 1A and [17]), it is possible that the lower MCM levels in mutant MEFs are entirely attributable to growth defects. To test this, we assessed the levels of nuclear MCM2 in S-phase cells by flow cytometry (Figure 1B). Although MCM2 levels in WT and Mcm4Chaos3/Chaos3 G1 nuclei were essentially the same (P = .65; t-test), mutant cells transitioned from G1 to S with 40% less nuclear MCM2 content than in WT (P,.02; t-test). The levels of nuclear MCM2 in WT decreased through S phase more sharply than in mutants, which transitioned Quantitative Mcm Perturbation in Mice to G2 with only ,23% less than controls (Figure 1B). This differential decline is apparent in the flow plots, where WT cells exhibit a greater downward slope in the S compartment (Figure 1B). The decreases in MCM2 from early to late S were 51% in WT and 38% in mutants. The MCM2 intra-S modulation phenomenon is also addressed in subsequent experiments. The marked differences in nuclear MCM2 concentration between actively proliferating (S-phase) WT and mutant cells indicates that a biochemical or regulatory basis, rather than a population skewing, underlies the differences in protein levels. Another possible explanation for the coordinated decrease in MCMs is that the mutant MCM4Chaos3 protein destabilizes the MCM2-7 hexamer and causes subsequent degradation of uncomplexed MCMs. Other groups reported that knockdown of Mcm2, Mcm3, or Mcm5 in human cells decreased the amount of other chromatin-bound MCMs [15–16], leading to a similar proposition that the cause was MCM2-7 hexamer destabilization [16]. If true, then we would expect mRNA levels to be unchanged in mutant cells. To test this, we performed quantitative RT-PCR (qRT-PCR) analysis of Mcm2-7, and several control housekeeping genes in Mcm4Chaos3/Chaos3 MEFs. Analysis of 5 littermate pairs of primary MEF cultures revealed that transcript levels for each of these genes in mutant cells was 51–65% of WT, similar to the protein decreases (Figure 1C). Levels of mRNA in the 7 housekeeping genes analyzed were not altered significantly (Figure 1C, right panel). This data suggest that either reduced MCM4 levels per se, or defects resulting from the Mcm4Chaos3 allele, cause a decrease in the levels of all Mcm mRNAs. Interestingly, the mRNA reduction appears to occur post-transcriptionally, a phenomenon that is currently under investigation (Chuang and Schimenti, unpublished observations). Decreased Mcm gene dosages cause elevated chromosomal instability and Mcm2-specific pan-decreases in Mcm mRNAs The Mcm4Chaos3 allele was identified in a forward genetic screen for mutations causing elevated micronuclei (MN) in red blood cells, an indicator of GIN [17]. While the altered MCM4Chaos3 protein may cause DNA replication errors as does a yeast allele engineered to contain the same amino acid change [19], it is also possible that the decrease in overall MCM levels in Mcm4Chaos3 mutants contributes to, or is primarily responsible for, elevated S- phase DNA damage and GIN as is seen in various cell culture models (see Introduction). To test this possibility, we generated mice from ES cells bearing gene trap insertions in Mcm2, Mcm3, Mcm6, and Mcm7 (Figure 2A; alleles are designated as Mcm#Gt). These gene traps are designed to disrupt gene expression by fusing the 59 end of the endogenous mRNA (via use of a splice acceptor) to a vector-encoded reporter, resulting in a fusion protein lacking the C-terminal portion of the endogenous (MCM) protein. As with a previously-reported Mcm4 gene trap [17], each of these alleles proved to be recessive embryonic lethal (Figure S1). Furthermore, each allele appeared to be a null, since mRNA levels in heterozygous MEF cultures were ,50% lower than WT controls (Figure 2B). To determine if heterozygosity for various Mcms caused pan-decreases in Mcm mRNA levels as does homozygosity for Mcm4Chaos3, mRNA levels for each of the Mcm2-7 genes were also quantified. Whereas Mcm2Gt/+ cells did show ,20% decreases in the other Mcms, the Mcm3, Mcm4, Mcm6 and Mcm7 gene trap alleles did not (Figure 2B). Thus, it appears that the marked Mcm pan-decreases in Mcm4Chaos3/Chaos3 cells are not due to decreased Mcm4 RNA per se, but rather a response to replication defects cause by the mutant protein. Notably, the pan Mcm2-7 downregulation in Mcm2Gt/+ cells is consistent with the observation that MCM7 is PLoS Genetics | www.plosgenetics.org 2 September 2010 | Volume 6 | Issue 9 | e1001110