br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments We are deeply thankful to V. Fossati and N. Lamas for helpful critical reading of the manuscript and to J. Goldman for generously providing the O4 antibody. We thank J.-Y. Li for providing access to wild-type mice for deriving mESCs; A. Collin for helping with karyotyping interpretation; C. Vignon for helping with the maintenance of the mESCs; A. Hammarberg, S. Giatrellis, and M. Toro for FACS advice; and J. Bigarreau for helping with graphic illustrations. We are also thankful to P.H. Jensen for constructive feedback during the editing of the manuscript. Human fibroblast cell lines were obtained from Coriell, the Cell Line and DNA Biobank from Patients affected by Genetic Diseases (Istituto G. Gaslini), and the Parkinson Institute Biobank (http://www.parkinsonbiobank.com), members of the Telethon Network of Genetic Biobanks (project GTB12001) funded by Telethon Italy. We are thankful to the Brain Bank at Columbia University team for providing us with the human postmortem tissue samples. This work was supported by the Strong Research Environment MultiPark (multidisciplinary research on Parkinson’s disease at Lund University) and through external grants to L.R. from the Jeanssons foundation; the Swedish Parkinson foundation (Parkinsonfonden); the Holger Crafoord foundation; the Segerfalk foundation; the Åke Wibergs foundation; the Greta och Johan Kocks foundation; and donations for science, medicine, and technology at Fysiografen in Lund. L.R. is a young investigator supported by MultiPark–Strategic Research Environment at Lund University, funded by the Swedish government, and partner of BrainStem–Stem Cell Center of Excellence in Neurology, funded by Innovation Fund Denmark.
Introduction It is essential for stem cells, especially embryonic stem ccr2 inhibitor (ESCs), to maintain genome integrity. A key aspect of this is to ensure the fidelity of DNA replication. In eukaryotic genomes, DNA replication initiates at thousands of origins. Origins are licensed prior to S phase, a process that involves the recruitment of licensing factors MCM2, 3, 4, 5, 6, and 7 as double heterohexamers onto DNA (Evrin et al., 2009; Remus et al., 2009). During S phase, each MCM2–7 complex can initiate replication by acting as a helicase to unwind double-stranded DNA ahead of DNA polymerases (Bochman and Schwacha, 2009). MCM2–7 complexes are loaded onto the genome in 5- to 20-fold excess to the number utilized to initiate DNA replication. The excess MCM2–7 complexes usually remain dormant, but they initiate back-up replication forks to rescue replication when primary forks are slowed or stalled; therefore, they are called dormant origins (DOs) (Doksani et al., 2009; Ge and Blow, 2010; Ge et al., 2007; Ibarra et al., 2008). Replication forks frequently stall, for example, when encountering tightly bound protein-DNA complexes, transcription machinery, repetitive sequences, or DNA lesions (Makovets et al., 2004; Mirkin and Mirkin, 2007). Prolonged fork stalling increases the probability of fork collapse and double strand breaks, which could lead to chromosomal re-arrangements and genomic instability (Lambert et al., 2005). As a safeguard mechanism, DOs provide the first line of defense against fork stalling (Blow and Ge, 2009). Chromosomal fragile sites, which are prone to breakage upon replication stress, are shown to have lower capacity to activate DOs (Letessier et al., 2011). Mice with reduced DOs show genomic instability, age-related dysfunction, and develop tumors (Kunnev et al., 2010; Pruitt et al., 2007; Shima et al., 2007). Importantly, congenital hypomorphic MCM4 defects have been found in humans, associated with various abnormalities and elevated genomic instability (Gineau et al., 2012; Hughes et al., 2012).
Results
Discussion We have demonstrated that ESCs recruit ∼2-fold more DOs onto the genome than NSPCs. Upon reduction of DOs, the self-renewal of ESCs is unaffected, whereas their differentiation including toward NSPCs is impaired. This is due to a further reduction of DOs in NSPCs, presumably below the threshold required to rescue the endogenous fork stalling during DNA replication (Figure 4F). As a result, DNA damage is accumulated and cell death incurs, eventually leading to impaired neurogenesis in the Mcm4 mice. ESCs have been shown to employ unique mechanisms to maintain a more-stable genome than somatic cells, including efficient DNA repair, elimination of damaged cells, antioxidant defense, and suppression of mutagenesis (Giachino et al., 2013). Our study adds a new dimension to these unique properties by showing that ESCs use more DOs to effectively protect their genomes from replication stress and ensure their genome integrity.