then washed twice with 10 ml TBS-TX (0.1% TX-100). 16106 nuclei were placed into 1.5ml tubes in 1ml TBS-TX buffer+1% BSA for 15 min at room temperature. The primary antibody (Rabbit anti-mouse MCM2) was added for 60 min, then secondary antibody (FITC goat anti-rabbit) was added for 60 min. Finally, the nuclei were stained with propidium iodide (PI), and RNAse treated (batches optimized empirically) for 30 mins. Immunolabeled nuclei were analyzed by flow cytometry (using a BD FACSCalibur cytometer with CellQuest software), exciting the PI and FITC with a 488nm laser. Generation and validation of mouse lines bearing mutant Mcm alleles ES cell lines containing gene trap insertions in Mcm genes were obtained from Bay Genomics [Mcm3 (RRR002), Mcm6 (YHD248), Mcm7 (YTA285)] or the Sanger Institute [Mcm2 (ABO178)]. The Mcm4 line was previously reported [17]. Allele names are abbreviated as, for example, Mcm3Gt instead of the full name Mcm3Gt(RRR002)Byg. All of the original ES cells were of strain 129 origin, and the alleles were backcrossed into C3HeB/FeJ for $4 generations. To identify the exact insertion sites of the gene trap vectors, a ‘‘primer walking’’ procedure was used. This involved priming PCR reactions with :1) a fixed vector primer, and 2) one of a series of primers series corresponding to the intron in which the vector presumably integrated. PCR products were then sequenced. Genotyping of gene-trap-bearing mice was performed either by PCR amplification of the neomycin resistance gene within the vector, or by using insertion-specific assays (Table S2). Western blot analysis Cytosolic and chromatin-bound protein was extracted as described [44]. Antibody binding was detected with a Pierce ECL kit. Band were quantified using NIH Image J software. Antibodies- aMCM2: ab31159 (Abcam); aMCM3: 4012 (Cell Signaling); aMCM4: ab4459 (Abcam); aMCM5: NB100-78261 (Novus); aMCM6: NB100-78262 (Novus); aMCM7: ab2360 (Abcam); aBeta-actin: A1978 (Sigma); aTBP: NB500-700 (Novus). Quantitative RT-PCR (qPCR) Total RNA from P1 MEFs was DNAse I treated, then cDNA was synthesized from 1 mg of total RNA using the Invitrogen References 1. Gilbert DM (2001) Making sense of eukaryotic DNA replication origins. Science 294: 96–100. 2. Blow JJ, Dutta A (2005) Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol 6: 476–486. 3. Lei M (2005) The MCM complex: its role in DNA replication and implications for cancer therapy. Curr Cancer Drug Targets 5: 365–380. 4. Forsburg SL (2004) Eukaryotic MCM proteins: beyond replication initiation. Microbiol Mol Biol Rev 68: 109–131. 5. Tye BK (1999) MCM proteins in DNA replication. Annu Rev Biochem 68: 649–686. 6. Moyer SE, Lewis PW, Botchan MR (2006) Isolation of the Cdc45/Mcm2-7/ GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 103: 10236–10241. 7. Labib K, Tercero JA, Diffley JF (2000) Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288: 1643–1647. 8. Bochman ML, Schwacha A (2008) The Mcm2-7 complex has in vitro helicase activity. Mol Cell 31: 287–293. 9. Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, et al. (2001) DNA replication licensing and human cell proliferation. J Cell Sci 114: 2027–2041. 10. Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prokhorova TA, et al. (2002) MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts. J Biol Chem 277: 33049–33057. Quantitative Mcm Perturbation in Mice SuperScript III ReverseTranscriptase kit with the supplied Olige- dT or random-hexamer primers. qPCR reactions were performed in triplicate on 1 ng or 10 ng of cDNA by using the SYBR power green RT-PCR Master kit (Applied Biosystems; 40 cycles at 95uC for 10 s and at 60uC for 1 min), and real-time detection was performed on an ABI PRISM 7300 and analyzed with Geneamp 5700 software. The specificity of the PCR amplification proce- dures was checked with a heat-dissociation step (from 60uC to 95uC) at the end of the run and by gel electrophoresis. Results were standardized to b-actin. The PCR primers are listed in Table S1. Supporting Information Figure S1 Homozygous lethality of Mcm gene trap alleles. Found at: doi:10.1371/journal.pgen.1001110.s001 (0.06 MB PDF) Figure S2 Synthetic lethal interactions of gene trap alleles. Found at: doi:10.1371/journal.pgen.1001110.s002 (0.07 MB PDF) Mcm4Chaos3/Chaos3 Figure S3 Histopathology of Mcm2Gt/+ tumors. Found at: doi:10.1371/journal.pgen.1001110.s003 (4.87 MB PDF) Figure S4 Rescue of viability by Mcm3 hemizygosity. Found at: doi:10.1371/journal.pgen.1001110.s004 (0.06 MB PDF) Table S1 Mcm4Chaos3/Chaos3 Mcm2Gt/+ tumor pathology. Found at: doi:10.1371/journal.pgen.1001110.s005 (0.08 MB PDF) Table S2 PCR primers. Found at: doi:10.1371/journal.pgen.1001110.s006 (0.04 MB PDF) Acknowledgments The authors thank B. Tye and S. Pruitt for helpful comments on the manuscript, and R. Munroe for generating chimeric mice. Author Contributions Conceived and designed the experiments: CHC JCS. Performed the experiments: CHC MDW CA. Analyzed the data: CHC MDW TS JCS. Wrote the paper: CHC TS JCS. 11. Woodward AM, Gohler T, Luciani MG, Oehlmann M, Ge X, et al. (2006) Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. J Cell Biol 173: 673–683. 12. Lei M, Kawasaki Y, Tye BK (1996) Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae. Mol Cell Biol 16: 5081–5090. 13. Crevel G, Hashimoto R, Vass S, Sherkow J, Yamaguchi M, et al. (2007) Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells. PLoS ONE 2: e833. 14. Young MR, Tye BK (1997) Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae. Mol Biol Cell 8: 1587–1601. 15. Ge X, Jackson DA, Blow JJ (2007) Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev 21: 3331–3341. 16. Ibarra A, Schwob E, Mendez J (2008) Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc Natl Acad Sci U S A 105: 8956–8961. 17. Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, et al. (2007) A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nat Genet 39: 93–98. 18. Fletcher RJ, Bishop BE, Leon RP, Sclafani RA, Ogata CM, et al. (2003) The structure and function of MCM from archaeal M. Thermoautotrophicum. Nat Struct Biol 10: 160–167. PLoS Genetics | www.plosgenetics.org 14 September 2010 | Volume 6 | Issue 9 | e1001110