Incremental Genetic Perturbations to MCM2-7 Expression and Subcellular Distribution Reveal Exquisite Sensitivity of Mice to DNA Replication Stress Chen-Hua Chuang, Marsha D. Wallace, Christian Abratte, Teresa Southard, John C. Schimenti* Department of Biomedical Sciences and Center for Vertebrate Genomics, Cornell University, Ithaca, New York, United States of America 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 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. Citation: Chuang C-H, Wallace MD, Abratte C, Southard T, Schimenti JC (2010) Incremental Genetic Perturbations to MCM2-7 Expression and Subcellular Distribution Reveal Exquisite Sensitivity of Mice to DNA Replication Stress. PLoS Genet 6(9): e1001110. doi:10.1371/journal.pgen.1001110 Editor: Wayne N. Frankel, The Jackson Laboratory, United States of America Received June 1, 2010; Accepted August 3, 2010; Published September 9, 2010 Copyright: 2010 Chuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by a Department of Defense Breast Cancer Program predoctoral fellowship BC083376 to CC, a fellowship slot on National Institutes of Health (NIH) training grant T31 GM7617 for MW, NIH grant R01GM45415 to JS, and a NYSTEM core grant to JS (http://stemcell.ny.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jcs92@cornell.edu Introduction In late mitosis to early G1 phase of the cell cycle, DNA replication origins are selected and bound by the hexameric origin recognition complex (ORC; [1]). ORC then recruits the initiation factors CDC6 and CDT1, which are required for loading MCM2- 7, thereby forming the ‘‘pre-replicative complex’’ (pre-RC). The formation of pre-RCs is termed origin ‘‘licensing’’ and this gives origins competency to initiate a single round of DNA synthesis before entering S phase. MCM2-7 is a hexamer of six distinct but structurally-related minichromosome maintenance (MCM) pro- teins (reviewed in [2–5]). In vivo and in vitro evidence indicates that the MCM2-7 complex is the replicative helicase [6–8]. MCM2-7 proteins are abundant in proliferating cells [9], and are bound to chromatin in amounts exceeding that which is present at active replication origins or required for complete DNA replication [10–14]. Although these and other studies showed that drastic decreases in MCMs are tolerated by dividing cells, there are certain deleterious consequences. In Xenopus extracts and mammalian cells, excess chromatin-bound MCM2-7 complexes occupy dormant or ‘‘backup’’ origins that are activated under conditions of replication stress, compensating for stalled or disrupted primary replication forks [11,15–16]. The depletion of these backup licensed origins was associated with elevated chromosomal instability and susceptibility to replication stress, factors that might predispose to cancer. In previous work, Shima et al found that a hypomorphic allele of mouse Mcm4 (Mcm4Chaos3) caused high levels of GIN and extreme mammary cancer susceptibility in the C3HeB/FeJ background [17]. This provided the first concrete evidence that endogenous mutations in replication licensing machinery may have a causative role in cancer development. The ethylnitrosourea (ENU)-induced Mcm4Chaos3 point mutation changed PHE to ILE at residue 345 (Phe345Ile). This amino acid is conserved across diverse eukaryotes and is important for interaction with other MCMs [18]. Budding yeast engineered to bear the orthologous mutation exhibit DNA replication defects and GIN [17,19]. Surprisingly, MEFs from Mcm4Chaos3 mice not only had reduced levels of MCM4, but also MCM7 [17], suggesting that the point mutation might destabilize the MCM2-7 complex. Subsequently, it was reported that mice containing 1/3 the normal level of MCM2 succumbed to lymphomas at a very young age, and had diverse stem cell proliferation defects [20]. These mice also had 27% reduced levels of MCM7 protein, and their cells exhibited decreased replication origin usage when under replication stress (treatment with hydroxyurea) conditions [21]. These studies imply PLoS Genetics | www.plosgenetics.org 1 September 2010 | Volume 6 | Issue 9 | e1001110