Chromosoma DOI 10.1007/s00412-014-0489-2 REVIEW Switch on the engine: how the eukaryotic replicative helicase MCM2–7 becomes activated Silvia Tognetti & Alberto Riera & Christian Speck Received: 20 July 2014 /Revised: 24 September 2014 /Accepted: 25 September 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract A crucial step during eukaryotic initiation of DNA cycle and the activation of the helicase in S-phase. replication is the correct loading and activation of the replicative Importantly, helicase loading is strictly inhibited in S-phase, DNA helicase, which ensures that each replication origin fires which guarantees that no piece of DNA is replicated more only once. Unregulated DNA helicase loading and activation, than once. During tumorigenesis, unregulated helicase load- as it occurs in cancer, can cause severe DNA damage and ing and activation occurs resulting in severe genomic insta- genomic instability. The essential mini-chromosome mainte- bility. This in part explains why cancerous cells are character- nance proteins 2–7(MCM2–7) represent the core of the eu- ized by high cell death rates. Nevertheless, the high mutation karyotic replicative helicase that is loaded at DNA replication rates also offer the surviving cells a chance to adapt quickly or origins during G1-phase of the cell cycle. The MCM2–7 acquire new functions. helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2–7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activa- DNA licensing tion, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure Initiation of DNA replication begins with the recruitment of genome integrity. Here, we review the current understanding of the replicative helicase to origin DNA. To allow efficient helicase activation; we focus on protein interactions during DNA synthesis, it is necessary to load multiple copies of the CMG formation, discuss structural changes during helicase replicative DNA helicase at hundreds of origins in activation, and outline similarities and differences of the pro- Saccharomyces cerevisiae or tens of thousands of origins in karyotic and eukaryotic helicase activation process. humans. This is a challenging task, since DNA accessibility inside the nucleus varies significantly depending on its posi- tion on the chromosome and chromatin composition (e.g., transcriptionally active or silent chromatin, telomeres or cen- Introduction tromeres, etc.). Eukaryotic cells have evolved a sophisticated machinery to achieve efficient helicase recruitment and load- DNA replication is a critical cellular process essential for ing. In S. cerevisiae eight protein factors make up the helicase stable maintenance of the genome prior to cell division. loading machine: the six-subunit origin recognition complex Cells have evolved a sophisticated regulatory network that (ORC1-6), Cdc6 and Cdt1 (Riera et al. 2014). During helicase allows efficient and controlled replication of the genome. This loading, also termed pre-replication complex (pre-RC) forma- involves loading of the replicative DNA helicase at many tion, two copies of the MCM2–7 helicase are loaded in a evenly spaced replication origins across the full length of the stable head-to-head double-hexamer around double-stranded chromosome in late M-phase to early G1-phase of the cell (ds) origin DNA (Evrin et al. 2009; Gambus et al. 2011; Remus et al. 2009). The reconstitution of budding yeast pre- : : * S. Tognetti A. Riera C. Speck ( ) RC formation with purified proteins has significantly ad- DNA Replication Group, Institute of Clinical Science, Imperial College, London W12 0NN, UK vanced our understanding of this vital reaction. Therefore, e-mail: [email protected] we will focus on describing the process in this organism. Chromosoma Pre-RC formation is initiated by ORC, which specifically hexamer is formed, it can slide on dsDNA in an ATP-hydrolysis binds to replication origins, and stays attached to chromatin independent manner, similarly to a ring on a string (Evrin et al. throughout the cell cycle (Bell and Stillman 1992; Liang and 2009; Remus et al. 2009). This may allow MCM2–7dispersal Stillman 1997). The ORC-DNA complex recruits Cdc6 in late away from replication origins, which is consistent with obser- M early G1 phase of the cell cycle to the replication origin vations that the MCM2–7 complex can be found at sites other (Weinreich et al. 1999). Binding of ORC and Cdc6 is an ATP- than replication forks (Laskey and Madine 2003). Moreover, dependent process (Speck et al. 2005) and involves interac- during pre-RC formation, multiple MCM2–7 double-hexamers tions between Cdc6 and the Orc1 and Orc2 subunits (Huo are loaded at replication origins. These additional copies func- et al. 2012). Then, ORC-Cdc6 recruits MCM2–7-Cdt1 to tion to establish new forks if there is a terminal fork collapse replication origins (Coleman et al. 1996; Donovan et al. due to severe DNA damage (McIntosh and Blow 2012). 1997; Evrin et al. 2009; Randell et al. 2006; Remus et al. Although MCM2–7 double-hexamer formation is crucial for 2009; Tanaka et al. 1997). In budding yeast, Cdt1 is required DNA replication, this complex has no DNA unwinding activity for the nuclear import of MCM2–7 (Tanaka and Diffley (Evrin et al. 2009; Remus et al. 2009) and needs to be activated 2002). Moreover, a Cdt1 interaction with Mcm6 alleviates for S-phase to start. an inhibitory activity in the C-terminus of Mcm6 (Fernandez-Cid et al. 2013), which in turn allows formation of the ORC-Cdc6-Cdt1-MCM2–7 (OCCM) complex that en- Helicase activation circles double-stranded DNA (Evrin et al. 2013; Samel et al. 2014; Sun et al. 2013). OCCM establishment induces Orc1 Eukaryotic helicase activation is best understood in the model and Cdc6 ATP hydrolysis and promotes Cdt1 release, organism S. cerevisiae, although the general principles are resulting in an ORC-Cdc6-MCM2–7(OCM)complex conserved across all eukaryotes. During the activation reac- (Fernandez-Cid et al. 2013; Randell et al. 2006). This OCM tion, an excess of 30 protein factors are assembled on origins complex is an intermediate of the pre-RC reaction and is, in to form a pre-initiation complex (pre-IC) (Gambus et al. 2006; contrast to the OCCM, able to recruit a second MCM2–7 Zou and Stillman 1998), which leads to the establishment of hexamer to the DNA. Once the second MCM2–7hexameris active replication forks. Two kinases are crucial for pre-IC recruited, a stable hexamer-hexamer interface is established formation, cyclin-dependent kinase (CDK) and Dbf4 depen- (Evrin et al. 2014), which leads to formation of a head-to-head dent kinase Cdc7 (DDK). Both promote complex assembly MCM2–7 double-hexamer (Riera et al. 2014). A summary of and coordinate the timing of replication initiation at hundreds the known protein-protein interactions involved in of origins (Siddiqui et al. 2013). Pre-IC formation culminates S. cerevisiae pre-RC formation can be found in Table 1. in the activation of the replicative helicase, which is composed The loading of the MCM2–7 double-hexamer at replication of MCM2–7 and two accessory factors Cdc45 and GINS that origins makes sense, as this complex is ideally suited to set up together form the active holo-enzyme, the Cdc45-MCM2–7- bidirectional replication forks in S-phase, with each fork GINS (CMG) complex (Costa et al. 2011;Gambusetal.2006; inheriting one MCM2–7 hexamer. Importantly, CDK is a Moyer et al. 2006). prime inhibitor of helicase loading in S-phase, which is essen- tial to circumvent re-replication (Nguyen et al. 2001). It was discovered recently that CDK-dependent phosphorylation of Overview of pre-IC formation ORC specifically affects the ATP-hydrolysis driven OCCM to OCM transition, indicating that pre-RC formation is not con- S. cerevisiae pre-IC formation is divided into two discrete trolled at the level of initial complex formation, but that OCM steps according to the requirements for DDK and CDK ki- formation is subject to a quality control mechanism nases. At the first step during late G1 phase, DDK activity (Fernandez-Cid et al. 2013; Frigola et al. 2013). All pre-RC promotes the association of Sld3, Sld7, and Cdc45 with rep- intermediates are sensitive to salt washes; however, the lication origins (Heller et al. 2011;Kamimuraetal.2001; MCM2–7 double-hexamer that encircles dsDNA is stable in Kanemaki and Labib 2006; Tanaka et al. 2011). At the second the presence of high salt (Donovan et al. 1997; Evrin et al. step in a CDK-dependent manner, Sld2, Dpb11, DNA poly- 2014; Remus et al. 2009). This stability of the MCM2–7 merase ε (Polε), and GINS form a pre-loading complex (pre- double-hexamer on chromatin is essential, as the loss of this LC) (Muramatsu et al. 2010) that associates with the origins complex from DNA during the G1-S transition would lead to via an Sld3-Dpb11 interaction (Heller et al. 2011;Masumoto inefficient DNA replication and genomic instability, since et al. 2002; Muramatsu et al. 2010;Tanakaetal.2007; reloading of the helicase is blocked during S-phase. However, Zegerman and Diffley 2007). As a consequence, the only now we are beginning to understand the molecular basis of MCM2–7 double-hexamer is transformed into the CMG com- the MCM2–7 double-hexamer stability (Sun et al.; Genes & plex, which functions as the active DNA helicase. Within the Development 2014). Interestingly, once the MCM2–7 double- pre-RC, MCM2–7 encircles dsDNA, but the CMG encircles Chromosoma Table 1 Summary of known protein–protein interactions involved in helicase activation Interaction Binding involves Method Reference pre-RC proteins ORC–Cdc6 Orc1 and Orc2, Cdc6 FL Two hybrid (Huo et al.