Incremental Genetic Perturbations to MCM2-7 Expression and Subcellular Distribution Reveal Exquisite Sensitivity of Mice to DNA Replication Stress

Abstract

Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4Chaos3 cancer susceptibility allele. Mcm4Chaos3/Chaos3 embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also “backup” origins that are recruited in times of replication stress to ensure complete replication of the genome.

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

“…To examine genomic copy number changes, we performed array comparative genomic hybridization (aCGH) on 12 Chaos3 tumors (nine Chaos3 mammary and three nonmammary), and two MMTVNeu mammary tumors. Chaos3 nonmammary tumors can be obtained by genetic perturbations or altering the strain background (Chuang et al 2010;Kawabata et al 2011b). Strikingly, the Chaos3 tumors exhibited highly recurrent CNAs in a small number of regions.…”

The MCM-associated protein MCM-BP is important for human nuclear morphology

“…It is presently unclear whether the increase in MCM protein levels itself could result in some of the phenotypes associated with MCM-BP depletion, as the role of the soluble pool of MCM proteins is currently unknown. However, it is interesting to note that a recent study in mice has shown that seemingly small decreases in MCM protein levels can affect MCM localization and result in replication stress (Chuang et al, 2010). It remains to be determined whether increases in MCM levels also have detrimental effects.…”

MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans

“…This is supported by the observation that the capsule appears to be thin adjacent to the regions of infiltration, further suggesting that over time the rate of infiltration exceeds the ability of the capsule to self-renew. Depletion of MCMs has been proposed to lead to stem cell defects in mice (7,18), with lower MCM2 levels associated with reduced numbers of stem cells in the subventricular zone, skeletal muscle, and intestinal crypts in adult mice. Similarly, the relatively specific impingement of the MCM4 defect on adrenal function may be a consequence of its effect on the growth of these mesenchymal stem/progenitor cells and their differentiation into steroidogenic cells.…”