tion or nuclear export. If WT nuclei have more dormant licensed origins than Chaos3 mutants, then WT cells would be expected show a greater loss of MCMs. The isolation of Mcm4Chaos3 provided the first demonstration that mutant alleles of essential replication licensing proteins can cause GIN and cancer [17]. Diploid budding yeast containing the same amino acid change in scMcm4 as the mouse Mcm4Chaos3 exhibited Rad9-dependent G2/M delay (Rad9 is a DNA damage checkpoint protein), elevated mitotic recombination, chromosome rearrangements, and intralocus mutations [19] (Li, X. and Tye, B., personal communication). One explanation for these outcomes is that the Chaos3 mutation impairs MCM4 biochemically in a manner leading to elevated replication fork defects, and that these defects lead to the GIN and cancer phenotypes. Alternatively, and/or in addition, the observed associated pan-reductions of MCMs in mouse cells [17] raised the possibility that decreased replication licensing might be the primary or ancillary cause for the mouse phenotypes. The subsequent finding that mice (Mcm2IRES-CreERT) containing ,1/3 the normal level of MCM2 had GIN and and cancer lent support for the idea that reductions in MCMs contribute to the Chaos3 phenotypes [20]. Although amounts of all MCMs were not investigated in Mcm2IRES-CreERT/IRES-CreERT mice, 65% reduc- tion of MCM2 caused a reduction of dormant replication origins in MEFs that were replication stressed by hydroxyurea [21]. In Mcm4Chaos3/Chaos3 mice, we hypothesize that in the context of Mcm2, 6 or 7 heterozygosity, which further reduces overall and chromatin-bound MCM levels below that already caused by Mcm4Chaos3 (measured to be ,20% of WT mRNA levels for Mcm2), MCMs are reduced to a degree that compromises cell proliferation. This then translates into the various developmental defects and increased cancer susceptibility we observed. Whatever the exact mechanistic cause of these phenotypes, it is clear that the phenotypes are related to reduction of one or more MCMs below a threshold level that is ,50%. The severe developmental consequences of MCM depletion in mice suggests that certain cell types in the developing embryo are highly sensitive to the effects of replicative stress, and/or that relatively minor cell growth perturbations of such cells are not well-tolerated in the context of complex, rapidly-occuring developmental events. The molecular basis for these phenotypes does not appear to be directly related to GIN, because whereas Mcm3 hemizygosity rescued several phenotypes, and delayed cancer latency in Mcm4Chaos3/Chaos3 mice, it did not concommitantly decrease MN. This suggests that phenotypes such as decreased proliferation and embryonic death are caused by genetically-induced replication stress, moreso (or in addition to) than GIN alone. Our genetic studies indicate that there is a quantitative MCM threshold required for embryonic viability, as demonstrated by the synthetic lethalities we observed when combining homozy- gosity of Mcm4Chaos3 with Mcm2Gt, Mcm6Gt or Mcm7Gt heterozy- gosity, but not in the heterozygous single mutants. Additionally, the Mcm4Chaos3/Gt genotype, which reduced MCM levels below 50%, caused embryonic and neonatal lethality [17]. Underscor- ing the exquisite sensitivity of whole animals to subtle perturbations in the DNA replication machinery were the remarkable phenotypic rescues (viability, growth, iPS efficiency, etc.) by Mcm3 hemizygosity. The decreased MCM dosage led to increases in S phase nuclear MCMs and chromatin-bound MCMs, presumably reflecting increased replication origin formation. The various single and compound mutants described here and elsewhere [20], which show that 50% reductions of any one MCM is well-tolerated but decreases of ,2/3 are not, supports the idea of a threshold effect, and suggests that the Quantitative Mcm Perturbation in Mice threshold lies somewhere between 1/3 and 1/2 of normal MCM levels (at least in the cases of MCM2, MCM6 and MCM7). These results also emphasize the importance of relevant physiological models, both in general and with respect to the MCMs. RNAi knockdown of MCM3 in human cells to ,3% normal levels was still compatible with normal short-term proliferation, although the cells had GIN and high sensitivity to replication stress [16]. It is doubtful such a drastic situation would be recapitulated in vivo (it would likely result in embryonic lethality as in Mcm3Gt/Gt mice). Nevertheless, it is noteworthy in that study that MCM3 depletion was better tolerated than knockdowns of any other member of the replicative helicase. The finding that reductions in MCM3 rescued MCM2/4/6 depletion phenotypes lends insight into dynamics and regulation of mammalian DNA replication. In budding yeast, MCMs shuttle between the nucleus and cytoplasm during the cell cycle. MCM2-7 multimers must be assembled in the cytoplasm before being imported into the nucleus during G1 phase [4]. The MCM2-7 importation is dependent upon synergistic nuclear localization signals (NLS) on Mcm2 and Mcm3 [22]. In order to prevent over- replication of the genome, MCMs are exported from the nucleus during S, G2 and M [4]. This export is dependent upon Mcm3, which has a nuclear export signal (NES) that is recognized by Cdc28 to promote MCM2-7 export in a Crm1-dependent manner [22]. In contrast to budding yeast, MCMs that have been studied (MCM2/3/7) are primarily nuclear-localized throughout the cell cycle in metazoans and in fission yeast [4]. Upon dissociation from chromatin during S phase, MCM2-7 complexes are reported to remain in the nucleus but are sequestered via attachement to the nuclear envelope or other nuclear structures [24,33–35]. Interest- ingly, mcm mutations in fission yeast that disrupt intact MCM2-7 heterohexamers triggers active redistribution of MCMs to the cytoplasm [36]. Additionally, re-distribution of MCMs between the cytoplasmic and nuclear compartments has been observed in hormonally-treated mouse uterine cells [25]. Our observations support the idea that intracellular re- distribution of MCMs also occurs in mammals, and that it is an important regulatory process. Staining of MCM2 in intact nuclei of normal NIH 3T3 fibroblasts and MEFs show a steady decline (but not elimination) as S phase progresses. Furthermore, it appears that the process of nuclear MCM2 elimination during S phase is regulated, since in situations of decreased MCMs (as in the Mcm4Chaos3/Chaos3 mutant), there is decreased loss of nuclear MCM2 during S phase. Three lines of experimentation implicate MCM3 as playing a key role in regulating intracellular MCM localization: 1) Rescue of reduced-MCM phenotypes by genetic reduction of MCM3; 2) Increased S-phase nuclear MCM2 by Mcm3 hemizygosity in MCM-depleted cells (Figure 7D); and increased chromatin-bound MCM2/4 by Mcm3 hemizygosity in MCM-depleted cells. Our data suggests that MCM3 acts as a negative regulator that prevents re-assembly or reloading of MCM complexes as they dissociate from DNA during replication. As described earlier, mouse and human MCM3 have predicted NESs in similar positions of their primary amino acid sequences as do the yeast genes. Thus, one explanation for these phenomena is that decreased MCM3 suppresses MCM2-7 nuclear export, which occurs normally and which may be accentuated by the Chaos3 mutation in a fashion analogous to mcm mutant fission yeast discussed above [36]. This would effectively increase the amounts of MCMs available for replication licensing. More work is required to determine if the rescue mechanism is indeed related to a decrease in MCMs export, as opposed to direct or indirect PLoS Genetics | www.plosgenetics.org 12 September 2010 | Volume 6 | Issue 9 | e1001110