How the Eukaryotic Replicative Helicase MCM2–7 Becomes Activated

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 position 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 essential 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. 2012) Orc6–Cdt1 Orc6 aa 1–185, 270–435; Cdt1 FL Purified proteins (Chen et al. 2007) Full length proteins Purified proteins (Chen et al. 2007) Cdc6–Mcm3 Full length proteins Purified proteins (Sun et al. 2013) Cdt1–MCM2–7Cdt1aa1–312, MCM2–7 FL Purified proteins (Fernandez-Cid et al. 2013) Cdt1 aa 306–604, MCM2–7 FL Purified proteins (Fernandez-Cid et al. 2013) Cdt1 aa 472–604, MCM2–7 FL Purified proteins (Takara and Bell 2011) pre-IC proteins MCM2–7–DDK Mcm4 and Cdc7 Purified proteins (Sheu and Stillman 2006) Mcm2 aa 204–278, DDK Purified proteins (Bruck and Kaplan 2009) Mcm2 and Dbf4 Purified proteins (Ramer et al. 2013) Mcm5 and Cdc7 Purified proteins (Ramer et al. 2013) MCM2–7–Sld3 Full length proteins Purified proteins (Bruck and Kaplan 2011a) Sld3–Sld7 Sld3 aa 60–120, Sld7 FL Two hybrid and purified proteins (Tanaka et al. 2011) Sld3–Cdc45 Full length proteins Two hybrid (Kamimura et al. 2001) Sld3 aa 150–430, Cdc45 FL Two hybrid (Tanaka et al. 2007) Full length proteins Two hybrid (Zegerman and Diffley 2010) Full length proteins Purified proteins (Bruck and Kaplan 2011a; Bruck and Kaplan 2013) Sld3–GINS Sld3 FL and Psf1 FL Two-Hybrid (Takayama et al. 2003) Cdc45–MCM2–7 Full length proteins Purified proteins (Bruck and Kaplan 2011a) Sld3–Dpb11 Sld3 aa 527–668, Dpb11 FL Two hybrid (Tanaka et al. 2007) Full length proteins Two hybrid (Zegerman and Diffley 2010) Full length proteins Purified proteins (Bruck and Kaplan 2011b) Full length proteins Purified proteins (Wang et al. 2012) Cdc45–GINS Full length proteins Purified proteins (Bruck and Kaplan 2011a) Cdc45–Rad53 Full length proteins Two hybrid and purified proteins (Aucher et al. 2010) Sld2–MCM2–7 Full length proteins Purified proteins (Bruck et al. 2011) Dpb11–Sld2 Full length proteins Two hybrid (Kamimura et al. 1998) Full length proteins Two hybrid (Tak et al. 2006) Dpb11–Polε Dpb11 FL and Dpb2 FL Two hybrid (Edwards et al. 2002) Dpb11–GINS Dpb11 FL and Psf1 FL Two hybrid (Takayama et al. 2003) Sld2–Polε Sld2 FL and Dpb2 FL Purified proteins (Muramatsu et al. 2010) Polε–GINS Dpb2 FL and Sld5 FL Purified proteins (Muramatsu et al. 2010) GINS–Ctf4 Full length proteins Purified proteins (Tanaka et al. 2009) Full length proteins Purified proteins (Gambus et al. 2009) Polα–Ctf4 Pol1 FL and Ctf4 FL Two hybrid (Zhou and Wang 2004) Full length proteins Two hybrid (Gambus et al. 2009) Mcm10–MCM2–7 Mcm10 FL and Mcm2 FL Two hybrid (Merchant et al. 1997) Mcm10 FL and Mcm2 FL Two hybrid (Lee et al. 2010) Mcm10 FL and Mcm3 FL Two hybrid (Liachko and Tye 2009) Mcm10 FL and Mcm6 FL Two hybrid (Merchant et al. 1997) Mcm10 FL and Mcm6 FL Two hybrid (van Deursen et al. 2012) single-stranded DNA (ssDNA) (Fu et al. 2011), indicating that changes during pre-IC formation, which must involve the MCM2–7 complex undergoes large conformational MCM2–7 ring opening. Once the CMG is formed, Mcm10 Chromosoma seems to activate the complex, since the CMG cannot unwind helicase is loaded straight on ssDNA (Arias-Palomo et al. DNA in the absence of Mcm10 and stays associated with the 2013; Speck and Messer 2001). As a consequence, the origin (Kanke et al. 2012; van Deursen et al. 2012; Watase helicase can immediately start unwinding the DNA in a et al. 2012). Finally, with the help of Ctf4, DNA polymerase α processive manner. Although this mechanism requires a very (Polα) is recruited (Gambus et al. 2009;Kangetal.2013; efficient system of inhibiting helicase loading at replication Simon et al. 2014). Once Polα binds, ssDNA synthesis is origins that have already fired, the regulation is more simple, initiated by producing RNA primers through the primase because most bacteria only have one single replication origin subunits of the polymerase prior to processive DNA synthesis. and one single chromosome (Skarstad and Katayama 2013). Polε primarily functions for leading strand DNA synthesis, Eukaryotic genomes are significantly more complex with while DNA polymerase δ (Polδ) serves for lagging strand many chromosomes and large numbers of replication origins. DNA synthesis (Kunkel and Burgers 2008). A summary of Here, helicase loading and helicase activation need to be kept the reactions leading to CMG formation is shown in Fig. 1, separate in order to maintain genomic stability (Diffley 2010). whereas the known direct protein-protein interactions in- The incorporation of the two events (helicase loading and volved in initiation of replication are listed in Table 1 and activation) into separate cell cycle phases allows for tight shown in Fig. 2. Both are based on work in S. cerevisiae. regulation of DNA replication. In consequence of this, eu- Additionally, a comparison of the replication factors nomen- karyotes developed a helicase loading mechanism that is clature in S. cerevisiae, Schizosaccharomyces pombe and fundamentally different from bacteria. It is imperative that in vertebrates is given in Table 2. eukaryotes the helicase is loaded in an inactive form on DNA (G1-phase), which is maintained on DNA until it is activated in the following cell cycle phase. Currently, we are only – The pre-RC to CMG transition—the known unknowns beginning to understand the structural changes in MCM2 7 that regulate its helicase activity during DNA loading in G1- Initiation of DNA replication in bacteria is fairly simple, the phase and helicase activation in S-phase (Sun et al.; Genes & DnaA protein unwinds origin DNA and then the replicative Development 2014). One important principle is that the MCM2–7 helicase is loaded onto dsDNA; in this configura- tion, helicase activity is likely inhibited. Consistently, no DNA unwinding was observed for the MCM2–7 double- hexamer (Evrin et al. 2009; Remus et al. 2009). However, this inherently inactive form of a helicase represents a major challenge for the cell, as the complex needs to be activated in S-phase. We know that in budding yeast, a number of protein factors are involved in this process, namely Sld2, Sld3, Sld7, Dpb11, Cdc45, GINS, Polε, RPA, and Mcm10, but their function is entirely unknown (Boos et al. 2012;Labib 2010; Muramatsu et al. 2010). Interestingly, most of these factors do not exist in bacteria and only some in archaea, consistent with the idea that eukaryotes have evolved a novel system of helicase activation. Nevertheless, the CMG structure and its functional analysis have revealed important clues. The CMG complex contains a single MCM2–7 hexamer that encircles single-stranded DNA (Costa et al. 2011; Gambus et al. 2006; Yardimci et al. 2010, 2012), indicating that CMG formation requires two rather complex reactions: 1) separation of the MCM2–7 double hexamer and 2) opening of the MCM2–7 ring, DNA melting and the extrusion of the lagging strand followed by ring closure around the leading strand. The order of these two events is unknown. Assuming that the Fig. 1 Schematic representation of the initiation of replication. In late G1-phase, DDK phosphorylates the MCM2–7 double hexamer, which MCM2–7 hexamer has only one DNA exit gate for ssDNA allows the recruitment of Sld3 and Cdc45 to replication origins. Subse- extrusion, we suggest two different models (Fig. 3): If both quently, in S-phase, Mcm10 and the pre-LC, consisting of Sld2, Dpb11, exit gates are aligned along the horizontal axis of the MCM2– ε Pol and GINS, associate with the origins in a CDK-dependent manner. 7 double-hexamer, then ring opening and ssDNA extrusion The complex formed (pre-IC) is competent for DNA unwinding and recruitment of Polα and Polδ, prior to replisome formation and DNA could occur prior to hexamer separation (Fig. 3, model A). synthesis Alternatively, if the DNA exit gates are offset to each other, Chromosoma

Fig. 2 Protein–protein interactions in helicase loading and activation. Schematic representation of the interactions involved in a helicase loading and b the activation process. Solid lines represent direct physical interactions whereas dashed lines indicate interactions identified by the two hybrid system

ring opening would be blocked by the hexamer-hexamer discovered in respect of their activation mechanism. In partic- interactions. Indeed, very recently, data have been obtained ular, we need to identify the MCM2–7 exit gate for ssDNA that support the model B (Sun et al.; Genes & Development extrusion and analyze how the double-hexamer splits prior to 2014)(Costaetal.2014). Accordingly, double-hexamer sep- CMG formation. One easy way of separating the hexamers aration would need to occur prior to ring opening (Fig. 3, would be turn on the helicase motor; since both hexamers are model B). However, in this case, it is not clear how each facing opposite directions, this could lead to dissociation from individual hexamer can be directed to the correct DNA strand each other. It is not known how the MCM2–7 ATPase is during strand extrusion. Although we are beginning to under- regulated within the double-hexamer. However, within the stand how the two MCM2–7 hexamers are arranged relative to CMG complex, the ATP-hydrolysis rate of MCM2–7ismuch each other within the double-hexamer (Sun et al.; Genes & higher than for the purified MCM2–7 hexamer (Ilves et al. Development 2014), (Costa et al. 2014), much needs to be 2010), suggesting that the binding of GINS or Cdc45 could

Table 2 Orthologs of replication factors S. cerevisiae S. pombe Metazoa Function ORC (Orc1-6) ORC (Orp1-6) ORC Origin Recognition Complex, pre-RC component Cdc6 Cdc18 Cdc6 ATP-binding protein, pre-RC component Cdt1 Cdt1 Cdt1 DNA replication licensing factor, pre-RC component MCM2-7 MCM2-7 MCM2-7 Replicative helicase, pre-RC and CMG component - - Geminin DNA licensing inhibitor ??GEMC1 Required for recruitment of Cdc45 to chromatin ??DUE-B Required for recruitment of Cdc45 to chromatin Sld3 Sld3 Treslin/Ticrr Required for helicase activation and pre-IC formation Sld7 ? MTBP? Non-essential factor, interacts with Sld3/Treslin Cdc45 Cdc45/Sna41 Cdc45 Required for pre-IC formation, CMG component Sld2 Drc1 RecQ4/ RecQL4? Required for helicase activation and pre-IC formation, pre-LC component Dpb11 Cut5/Rad4 TopBP1/Cut5/Mus101 Loads Pol and GINS into pre-IC, pre-LC component GINS GINS GINS CMG and pre-LC component Pol Pol Pol Leading strand polymerase, pre-LC component MCM10 MCM10 MCM10 Required for CMG activation RPA RPA RPA Single-stranded DNA binding protein Ctf4 Mcl1 AND-1 Required for sister chromatid cohesion; interacts with Polα PCNA PCNA PCNA Sliding clamp of DNA polymerases Kinases involved in DNA replication Dbf4 Dfp1/Him1 Dbf4 DDK regulatory subunit Cdc7 Hsk1 Cdc7 DDK catalytic subunit Clb5 Cig2 Cyclin E Cyclin involved in S-phase, CDK regulatory subunit Cdc28 Cdc2 Cdc2/Cdk2 CDK catalytic subunit Mec1 Rad3 ATR Genome integrity checkpoint protein kinase Rad53 Cds1 Chk2 DNA damage response protein kinase The replicative factors that associate with origins in the absence of CDK are shown in gray; the factors that associate in a DDK-dependent manner are shown in red and in purple are shown the factors that require both DDK and CDK. No color is shown whenever the kinase requirement has not been directly determined. The question mark indicates that it is unknown if the homologue protein is present in the relevantorganism. The question mark followed by a protein name indicates that it is unclear if the named protein is thetrue homologue Chromosoma

MCM2–7 complex, touching the Mcm2, Mcm5, and Mcm3 subunits. However, the structure of MCM2–7 in the CMG complex appears to differ from the structure of the MCM2–7 hexamer on its own. D. melanogaster MCM2–7 assumes two different conformations: a notched planar form and a gapped spiral form. In each case, the MCM2–7 ring is broken, which was not observed with the CMG (Costa et al. 2011). This indicates that the purified MCM2–7 hexamer is unstable and that the binding of Cdc45 and GINS to MCM2–7promotes stabilization of the complex, which seems to be important for Fig. 3 Schematic representation of strand extrusion models. The double- its function. Additionally, it was found that the binding of hexamer is loaded onto dsDNA. Model A exit gates are aligned along the Cdc45 and GINS to MCM2–7 generates a large channel horizontal axis of the MCM2–7 double-hexamer. In this case, ring open- through the CMG complex that stretches from the centre of ing and ssDNA extrusion could occur prior to hexamer separation. Model the MCM2–7 pore to the inner surface of the Cdc45/GINS B DNA exit gates are offset from each other; therefore, ring opening is blocked by the hexamer–hexamer interactions. In this case, double- proteins. Furthermore, within the CMG, Cdc45 and GINS hexamer separation would need to occur prior to ring opening make extensive contact with each other, both seen in the EM structure and reported in a protein-protein interaction analysis turn on the ATPase motor of the helicase. Alternatively, a (Costa et al. 2011; Ilves et al. 2010;Imetal.2009). One side structural change in the double-hexamer interface, which is of Cdc45 interacts with the N-terminal α-helical domain of composed of the MCM2–7 N-terminal domains, could induce Psf2, while the other side contacts the N-terminus of Mcm2. the separation reaction. Indeed, DDK phosphorylates the On the other hand, GINS seems to interact with Mcm5. The MCM2–7 N-termini (Randell et al. 2010; Sheu and Stillman structure of the CMG suggests that the complex cannot asso- 2006); but it was recently found that this does not lead to ciate with dsDNA, as the central pore is not wide enough double-hexamer separation (Sun et al.; Genes & Development (Costa et al. 2011). Indeed, a biochemical analysis of the 2014) (On et al. 2014). Moreover, the activation of the CMG, using strand-specific DNA roadblocks and Xenopus helicase motor could also be linked to initial strand separation egg extracts suggested that the CMG is a 3′ to 5′ ssDNA (DNA unwinding). Alternatively, Mcm10 and Sld2 could translocase (ssDNA passing through the central channel), participate in this process as well, as they are reported to be suggesting that the helicase unwinds DNA via "steric exclu- ssDNA binding proteins (Kanter and Kaplan 2011; Warren sion" (Fu et al. 2011). Moreover, during DNA damage, GINS et al. 2008). If these factors participate in MCM2–7 ring can temporarily dissociate from the CMG complex, indicating opening and how this is coordinated with strand separation that the complex is more dynamic than previously assumed is not known. In conclusion, it remains a future challenge to (Hashimoto et al. 2012). understand how the known pre-IC proteins promote helicase In contrast to bacteria, where a hexameric helicase unwinds activation; at present, the unknowns far outweigh the knowns. DNA, eukaryotes have evolved a more complex holo- helicase, which contains Cdc45 and the four-subunit GINS complex in addition to the MCM2–7 hexamer. However, it The Cdc45-MCM2–7-GINS complex remains to be seen how these helicase accessory factors contribute to processive DNA unwinding, and what the path is of MCM2–7 represents the core of the eukaryotic helicase, but both DNA strands through the CMG complex. Importantly, this complex has very little helicase activity of its own models that address these issues can be generated with current (Bochman and Schwacha 2008;Ishimi1997; Lee and data. For example, it was reported that Cdc45 interacts tightly Hurwitz 2000). However, once Cdc45 and GINS are associ- with ssDNA, both in budding yeast (Bruck and Kaplan 2013) ated with MCM2–7, the complex acquires strong helicase and humans (Szambowska et al. 2014). Then again, human activity (Ilves et al. 2010). Some structural information on Cdc45 seems to have a preference for 3′-protruding strands the CMG complex has been obtained, giving insights into the and single-/double-strand DNA junctions, and the protein organization and function of the helicase. Moreover, as appears to be able to slide on DNA with 3′–5′ polarity MCM2–7, Cdc45 and GINS are highly conserved, data ob- (Szambowska et al. 2014). Additionally, a structural similarity tained from various systems can be integrated, resulting in a was found between Cdc45 and RecJ, a prokaryotic ssDNA more comprehensive understanding of the complex. The low- exonuclease (Krastanova et al. 2012; Sanchez-Pulido and resolution three-dimensional reconstruction of the Drosophila Ponting 2011). These findings indicate that Cdc45 interacts melanogaster CMG identified the arrangement of the proteins with ssDNA. Indeed, Szambowska and colleagues proposed (Costa et al. 2011): the complex assumes a planar form, with that Cdc45 in the CMG could act as a wedge for the separation Cdc45 and GINS binding to the side of a closed circular of the two strands (Szambowska et al. 2014). However, in the Chromosoma context of the CMG structure (Costa et al. 2011, 2014), Cdc45 residues in MCM2–7 did not have an effect on Cdc45 associ- appears distal to the site of DNA unwinding, arguing that ation with origins, but surprisingly impacted on GINS and Cdc45 could guide ssDNA to the polymerase δ or another RPA association. Additionally, the models are consistent with associated factor. Based on these models, only a restricted a recent electron microscopy analysis of the CMG complex, path of DNA is possible through the CMG complex. One which indicates that the leading DNA strand passes through option is that the leading strand of DNA passes through the the MCM2–7 central channel (Costa et al. 2014). central channel of MCM2–7, whereas the lagging strand Nevertheless, more studies will be required to validate/ inserts in the lateral channel produced between the N- invalidate these speculative models and to understand fully terminal and C-terminal parts in the Mcm2/Mcm5 interface, how the two strands of DNA pass through the CMG complex. possibly contacting at this point Cdc45 or the Cdc45/GINS interface (Fig. 4, model A). The other option is that the lagging strand makes limited contacts with MCM2–7 on its outer C- DDK-dependent regulation of DNA replication terminal surface and additional major contacts with Cdc45, in S. cerevisiae potentially involving a ssDNA binding groove (Costa et al. 2014;Krastanovaetal.2012; Sanchez-Pulido and Ponting Helicase activation is initiated by DDK, which targets the 2011), and GINS (Fig. 4, model B). Both models are consis- MCM2–7 double-hexamer. This reaction involves recruitment tent with a recent analysis of the DNA binding properties of an of the kinase to replication origins, which begins already in the archaeal MCM homolog and budding yeast MCM2–7. The G1 phase of the cell cycle (Katou et al. 2006; Natsume et al. study revealed that residues on the surface of the N-terminal 2013). Here, Dbf4 interacts with Mcm2 (Ramer et al. 2013), MCM2–7 OB-fold are crucial for interaction with ssDNA of whereas Cdc7 associates with both Mcm4 and Mcm5 (Ramer the leading strand (Froelich et al. 2014), consistent with the et al. 2013; Sheu and Stillman 2006). Thus, several docking idea that MCM2–7 encircles ssDNA. Mutations of these sites for DDK are available within the helicase. However, as these sites are spread across a large surface of the MCM2–7 complex, it seems possible that multiple DDK complexes are operating in the context of the MCM2–7doublehexamer. Purified DDK was found to phosphorylate the Mcm2, Mcm4, and Mcm6 subunits, with strong prevalence for MCM2–7 in its double-hexameric form (Sun et al.; Genes & Development 2014)(Francisetal.2009). DDK targets se- quences for phosphorylation characterized by an acidic residue at the +1 position, which can be either an acidic amino acid or a negative charge provided by a phosphoserine or phosphothreonine (Charych et al. 2008; Cho et al. 2006; Montagnoli et al. 2006). Indeed, both CDK and the checkpoint kinase Mec1 phosphorylate chromatin-associated MCM2–7, generating priming sites for DDK-dependent phosphorylation, which contributes to efficient helicase activation and genomic stability (Randell et al. 2010). It is likely that DDK has a large number of targets in the cell, but for budding yeast, it was shown that MCM2–7 is the essential target of the kinase, as mutations in subunits of the helicase can bypass the DDK requirement of the cell (Hardy et al. 1997;Sheuand Stillman 2010). In particular the Mcm4 N-terminus contains a domain with an inhibitory activity that becomes alleviated upon phosphorylation (Sheu and Stillman 2010). Then again, Fig. 4 Models showing the path of DNA through the CMG. C-terminal a point mutation in the N-terminus of Mcm5 also promotes – view of MCM2 7 in complex with Cdc45 and GINS. a The leading DDK-independent DNA replication (Hardy et al. 1997). The strand passes right through the central channel of the CMG, whereas the lagging strand inserts in the lateral channel at the Mcm2/Mcm5 interface, structural change associated with DDK-dependent phosphor- contacting at this point Cdc45 or the Cdc45/GINS interface. b The ylation of the MCM2–7 double-hexamer is unclear, although leading strand passes through the central channel of the CMG, while it has been shown recently that the change does not involve the lagging strand makes strong contacts with a ssDNA binding groove of – – separation of the MCM2 7 double-hexamer (On et al. 2014). Cdc45 and minor contacts with MCM2 7onitsouterC-terminalsurface – and GINS. The EM map of the CMG complex is based on EMDB-1832 On the other hand, DDK phosphorylation of MCM2 7may (Costa et al. 2011) generate new protein binding sites, because Sld3, Sld7, and Chromosoma

Cdc45 only bind to origins upon DDK activation (Heller et al. phosphorylates Orc2 and Orc6 (Nguyen et al. 2001), which in 2011; Natsume et al. 2013; Tanaka et al. 2011). To establish turn inhibits the interaction of ORC with Cdt1 (Chen and Bell temporal control of DNA replication the activity of DDK must 2011; Fernandez-Cid et al. 2013; Frigola et al. 2013). Second, be regulated. The Cdc7 kinase subunit is stable during the cell S-CDK-dependent phosphorylation of Cdc6 induces its deg- cycle; however, the regulatory Dbf4 subunit becomes radation by the SCF complex (Drury et al. 1997;Elsasseretal. ubiquitinated by the anaphase promoting complex/ 1999; Perkins et al. 2001). Third, phosphorylation of Mcm3 cyclosome (APC/C) and targeted for proteasome mediated results in MCM2–7/Cdt1 export from the nucleus (Labib et al. degradation during late mitosis and G1-phase of the cell cycle 1999;Likuetal.2005;Nguyenetal.2001). All these redun- (Ferreira et al. 2000; Oshiro et al. 1999;Peters2006; dant mechanisms prevent pre-RC formation during the G1–S Weinreich and Stillman 1999). Subsequently, phosphorylation transition, ensuring that no origin is being licensed again after of the APC/C activator protein Cdh1 by G1-CDKs prevents it has been already fired. Cdh1 from binding and activating the APC/C, thereby allowing S-phase kinases to accumulate (Jaspersen et al. Regulation of pre-IC formation 1999; Zachariae et al. 1998). This regulatory loop guarantees that Dbf4 activity is kept low during G1 phase of the cell The most important role of S-CDK is to promote initiation of cycle. However, it does not explain the regulation of the DNA replication. It was found that Sld2 and Sld3 are the two replication timing of individual chromosomal domains. essential targets of CDK in budding yeast (Masumoto et al. Several recent studies showed that DDK is modulated in a 2002;Tanakaetal.2007; Zegerman and Diffley 2007). chromosome loci specific form; for example, the Ctf19 kineto- However, it remains unknown whether this is also true for chore complex can recruit Dbf4 to the kinetochores in telophase higher eukaryotes. It was demonstrated that S-CDK phosphor- to promote early DNA replication in S-phase (Natsume et al. ylates Sld2 and Sld3 to induce interactions with Dpb11. 2013). Then again, origins that fire late during S-phase, e.g., Moreover, Dpb11 contains two pairs of BRCA1 C-terminus telomeres, have been shown to contain a negative regulator of (BRCT) domains, which have phosphopeptide-binding activ- DDK activity. The Rap1-interacting factor 1 (Rif1) protein ity, and are essential for Sld2 and Sld3 binding. The N- binds to late origins and recruits protein phosphatase 1 (PP1), terminal pair of BRCT domains bind Sld3-P and the C- which facilitates the dephosphorylation of the MCM2–7com- terminal pair of BRCT domains bind Sld2-P, forming a tri- plex and blocks helicase activation (Dave et al. 2014;Hiraga meric complex. On the other hand, it has been shown recently et al. 2014; Mattarocci et al. 2014; Renard-Guillet et al. 2014). that a flexible linker in between these two pairs of BRCT domains binds GINS and that this is important for efficient replication (Tanaka et al. 2013). In particular, the binding of CDK-dependent regulation of DNA replication Sld2-P to Dpb11 is crucial for the formation of a pre-loading in S. cerevisiae complex (pre-LC), which consists of Sld2-P, Dpb11, GINS and Polε, and functions for GINS recruitment to the replica- The eukaryotic cell cycle is controlled by the activity of tion origin and CMG formation (Muramatsu et al. 2010). CDKs, a family of threonine-serine protein kinases that phos- Importantly, Sld2 becomes phosphorylated on multiple sites; phorylate specific target proteins. The catalytic subunit of the however, the phosphorylation of Thr84 is essential for Dpb11 kinase is regulated by cell cycle-dependent association with a binding (Tak et al. 2006). Thr84 phosphorylation requires pre- regulatory subunit, a cyclin, whose expression and/or stability phosphorylation of several other sites in Sld2; therefore, is also cell cycle dependent. Association of a cyclin with CDK Thr84 becomes only phosphorylated at high CDK activity. controls specific cell cycle transitions. For instance, G1-CDK This regulatory principle could be important, as it allows promotes S-phase entry, S-CDK inhibits origins licensing and complete inhibition of pre-RC formation at low CDK concen- promotes DNA replication, and finally, M-CDK regulates trations, prior to helicase activation at high CDK concentra- chromosome segregation during mitosis (Humphrey and tions. Moreover, hierarchical multisite-phosphorylation is pre- Pearce 2005). Similarly to DDK, the S-CDK concentration dicted to allow a switch-like activation of Sld2 (Brummer is regulated by APC/C-mediated degradation and therefore et al. 2010). However, how switch-like Sld2 activation is follows a similar dynamic as described above for DDK (Ang coordinated with the replication timing program of the cell is and Wade Harper 2005). currently unclear. Remarkably, simultaneous bypass of the CDK and DDK Regulation of pre-RC formation pathways in G1 phase, using a Sld3–Dpb11 fusion (SD fusion), overexpression of a phospho-mimetic Sld2-T84D mu- Relatively low levels of S-CDK are sufficient to inhibit tation and an mcm4Δ74–174 allele allows extensive DNA MCM2–7 loading at replication origins. For tight control, synthesis in α-factor arrested G1 cells (Sheu and Stillman multiple pre-RC proteins become modified: firstly, the kinase 2010). Importantly, in case of DNA damage, Rad53 functions Chromosoma to inhibit both CDK- and DDK-dependent activation path- it is required for its association with origins (Im et al. 2009; ways. Rad53 acts on DDK directly by phosphorylating Dbf4, Kumagai et al. 2011; Van Hatten et al. 2002). Additionally, it whereas the CDK pathway is blocked by Rad53-mediated was recently suggested that the central domain of Sld3, con- phosphorylation of the essential CDK substrate, Sld3 (Duch taining the essential binding site for Cdc45, is conserved in the et al. 2011; Lopez-Mosqueda et al. 2010; Zegerman and Sld3/Treslin family of proteins (Itou et al. 2014). Furthermore, Diffley 2010). This regulatory principle inactivates DDK, human and Xenopus Treslin/Ticrr/Sld3 bind the N-terminal but allows CDK to remain active during S phase in the BRCT domain of TopBP1/Dpb11 and is phosphorylated by presence of DNA damage, which is crucial to prevent re- CDK on the same conserved sites (Boos et al. 2011;Kumagai licensing of origins that have already fired. et al. 2011). Also, the human checkpoint kinase 1 (Chk1) can reduce the interaction of Treslin/Sld3 with TopBP1/Dpb11, in the same way to what was observed for Rad53 regulation of the interaction between Sld3 and Dpb11 in budding yeast Differential regulation of pre-IC formation in S. pombe (Boos et al. 2011; Kumagai et al. 2011; Lopez-Mosqueda and vertebrates et al. 2010;ZegermanandDiffley2010), indicating that even though their sequence has diverged, Treslin and Sld3 function The core factors of the replicative helicase are conserved in an identical manner. between archaea and eukaryotes (MCM, Cdc45, GINS), while Likewise, RecQ4, has been suggested as the homolog of the pre-IC proteins (Dpb11, Sld2, Sld7, Sld3, Mcm10, Ctf4) Sld2, despite the fact that the two proteins have very limited have no apparent ortholog in bacteria and are not required for sequence conservation. The two proteins share only a weak viral DNA replication. Additionally, the latter factors have similarity in the N-terminal portion, but this region is essential evolved in higher eukaryotes and have acquired new protein for DNA replication in Xenopus (Matsuno et al. 2006; domains or functions (Tanaka and Araki 2013). Sangrithi et al. 2005). Moreover, RecQ4 association with S. pombe and S. cerevisiae helicase activation factors have origins is TopBP1/Dpb11 and pre-RC dependent. However, a fairly high sequence similarity, though their regulation is RecQ4 differs from Sld2 due to the following functional partially different. In S. pombe, DDK is required for Sld3 characteristics: unlike Sld2, RecQ4 contains a C-terminal recruitment to origins, but Sld3 association, other than in helicase domain and is not required for TopBP1/Dpb11, budding yeast, is Sna41/Cdc45 independent. Instead, Sna41/ Cdc45, or GINS interaction with origin DNA. Furthermore, Cdc45 associates with chromatin in a CDK-dependent manner RecQ4 and TopBP1/Dpb11 can bind in the absence of CDK (Fukuura et al. 2011; Yamada et al. 2004). Nevertheless, it was phosphorylation, which is essential for the Sld2-Dpb11 inter- found in S. pombe that the Sld3-origin interaction is essential action (Matsuno et al. 2006; Sangrithi et al. 2005), similarly to for the subsequent recruitment of Cut5/Dpb11, Drc1/Sld2, what observed for the interaction between S. pombe Sld3 and Sna41/Cdc45, and GINS (Yabuuchi et al. 2006). Similarly Cut5/Dpb11. Additionally, unlike Sld2, RecQ4 is not needed as in budding yeast, DDK is essential for the process of for Cdc45 and GINS binding to chromatin (Matsuno et al. helicase activation; though, in fission yeast DDK-bypass mu- 2006; Sangrithi et al. 2005), despite its requirement for repli- tants in the MCM2–7 helicase have not been identified. cation initiation. Finally, unlike Sld2, Xenopus RecQ4 binds However, the deletion of Rif1 (discussed in the DDK section) to Mcm10 and associates with the CMG in a Mcm10- or Mrc1, which encodes a protein that is required for mainte- dependent manner (Xu et al. 2009). In summary, although nance of replication fork integrity, can bypass the DDK re- RecQ4 and Sld2 share some similarities, it is still an open quirement in S. pombe (Hayano et al. 2012; Matsumoto et al. question if RecQ4 is the functional homolog of Sld2. 2011). Moreover, S. pombe CDK facilitates the interaction of TopBP1 is thought to be the functional homolog of Dpb11 Drc1/Sld2 and Sld3 with Cut5/Dpb11, as demonstrated in in vertebrates, as both proteins share important structural budding yeast (Fukuura et al. 2011; Nakajima and Masukata similarities (Garcia et al. 2005;Rappasetal.2011; Tanaka 2002; Noguchi et al. 2002;Sakaetal.1994), but then again, and Araki 2013). However, some differences have been ob- the CDK-dependent phosphorylation of Sld3 in S. pombe was served as well. Similarly to what is known for lower eukary- found to be less important for its interaction with Cut5/Dpb11 otes, the association of TopBP1/Dpb11 with chromatin is than in S. cerevisiae, probably because the Drc1/Sld2 interac- CDK dependent. Yet, other than in yeast, this interaction is tion with Cut5/Dpb11 stabilizes the trimeric Drc1/Sld2-Cut5/ modulated by CDK concentration. At low CDK concentra- Dpb11-Sld3 complex (Fukuura et al. 2011). tion,XenopusTopBP1/Dpb11 is recruited to chromatin via an In vertebrates, the predicted homologs of Sld3, Sld2, and interaction with ORC and at high CDK concentrations via a Dbp11 are Treslin/Ticrr, RecQ4 and TopBP1, respectively, but pre-RC interaction. Even the recruitment at low CDK concen- their amino acid and domain organization has diverged sig- tration is sufficient for DNA replication (Hashimoto and nificantly (Tanaka and Araki 2013). As in budding yeast, Takisawa 2003; Van Hatten et al. 2002). Moreover, Xenopus human and Xenopus Treslin/Ticrr/Sld3 can bind Cdc45, and TopBP1/Dpb11 and Cdc45 interact directly (Schmidt et al. Chromosoma

2008), which is probably important, since Cdc45 association assembly seem to interact in a cooperative manner during with chromatin requires TopBP1/Dpb11 (Kumagai et al. pre-IC assembly. Although the functional analysis of DDK 2011; Van Hatten et al. 2002). Vertebrate TopBP1 and and CDK kinases on pre-IC formation have allowed the S. cerevisiae Dpb11 contain BRCT domains of variable num- identification of a 2-step mechanism in complex assembly, ber (Garcia et al. 2005;Rappasetal.2011). Nevertheless, in the key question for years to come is the following: what is the Xenopus, the first two BRCT repeats of TopBP1/Dpb11 are function of these pre-IC proteins beyond their role in complex involved in Treslin/Sld3 binding and are essential for replica- assembly? In particular, we need to identify how these pro- tion (Kumagai et al. 2010). Additional BRCT domains of teins affect the MCM2–7 double-hexamer structure, MCM2– TopBP1, which do not exist in S. cerevisiae Dpb11, function 7 interaction with DNA and its helicase activity. Moreover, it in checkpoint control of DNA replication (Garcia et al. 2005), will be crucial to understand how the CMG complex activity indicating that this function has become more important for is regulated during DNA synthesis. Answering these ques- vertebrates. tions will require integrated approaches and new assays, spe- Homologs for Mcm10 and Polε have also been identified cifically investigating the functions of each individual com- in vertebrates. Budding and fission yeast Mcm10 is involved ponent during pre-IC formation. in activation of the CMG for processive DNA unwinding (Kanke et al. 2012; van Deursen et al. 2012; Watase et al. Acknowledgments We would like to thank A. Okorokov, C. Herrera, 2012), while human and Xenopus Mcm10 functions in the Y. Javadi and F. Pisani for comments on the manuscript, and Imperial assembly of the CMG (Di Perna et al. 2013;Imetal.2009; College and the MRC for funding. Wohlschlegel et al. 2002) together with Ctf4 and RecQ4 (Im et al. 2009). Additionally, an interaction between human TopBP1/Dpb11 and Polε has been reported (Makiniemi et al. 2001), suggesting that a counterpart of the pre-LC may also exist in metazoans. References Among all the replication factors involved in helicase activation in budding yeast, only Sld7 is currently lacking Ang XL, Wade Harper J (2005) SCF-mediated protein degradation and homologs in higher eukaryotes. However, very recently, cell cycle control. Oncogene 24:2860–2870. doi:10.1038/sj.onc. 1208614 MTBP (MDM two binding protein) has been proposed as Arias-Palomo E, O'Shea VL, Hood IV, Berger JM (2013) The bacterial the functional homolog of Sld7 in human cells, although their DnaC helicase loader is a DnaB ring breaker. Cell 153:438–448. doi: amino-acid sequence and organization is quite different. 10.1016/j.cell.2013.03.006 MTBP is reported to interact with Treslin/TICRR throughout Aucher W et al (2010) A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteo- the cell cycle, and its depletion inhibits DNA replication by mic data. Mol Cell Proteomics 9:2745–2759. doi:10.1074/mcp. preventing assembly of the CMG prior to origin firing (Boos M110.003319 et al. 2013). Moreover, two metazoan factors, GEMC1 and Balestrini A, Cosentino C, Errico A, Garner E, Costanzo V (2010) DUE-B (Balestrini et al. 2010; Chowdhury et al. 2010)bothof GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication. Nat Cell Biol 12:484–491 which bind to TopBP1 and Cdc45, are required in Xenopus for Bell SP, Stillman B (1992) ATP-dependent recognition of eukaryotic the recruitment of Cdc45 to chromatin. However, both factors origins of DNA replication by a multiprotein complex. Nature have no homolog in lower eukaryotes, indicating that activa- 357:128–134. doi:10.1038/357128a0 tion of the replicative helicase is more complex in vertebrates Bochman ML, Schwacha A (2008) The Mcm2-7 complex has in vitro helicase activity. Mol Cell 31:287–293 than in yeast. Nevertheless, metazoan DDK and CDK, simi- Boos D, Sanchez-Pulido L, Rappas M, Pearl LH, Oliver AW, Ponting CP, larly as in S. cerevisiae, regulate pre-IC formation, with DDK Diffley JF (2011) Regulation of DNA replication through Sld3- acting prior to CDK, and DDK-dependent phosphorylation of Dpb11 interaction is conserved from yeast to humans. Curr Biol MCM2–7(Masaietal.2000) facilitating Cdc45 binding of 21:1152–1157 chromatin (Masai et al. 2006), indicating that the overriding Boos D, Frigola J, Diffley JF (2012) Activation of the replicative DNA helicase: breaking up is hard to do. Curr Opin Cell Biol 24:423–430. DNA replication concepts are conserved between lower and doi:10.1016/j.ceb.2012.01.011 higher eukaryotes. Boos D, Yekezare M, Diffley JF (2013) Identification of a heteromeric complex that promotes DNA replication origin firing in human cells. Science 340:981–984. doi:10.1126/science.1237448 Bruck I, Kanter DM, Kaplan DL (2011) Enabling association of the GINS Outlook protein tetramer with the mini chromosome maintenance (Mcm)2-7 protein complex by phosphorylated Sld2 protein and single-stranded In budding yeast, all essential proteins for helicase activation origin DNA. J Biol Chem 286:36414–36426 have been identified. The same is not the case for the human Bruck I, Kaplan D (2009) Dbf4-Cdc7 phosphorylation of Mcm2 is required for cell growth. J Biol Chem 284:28823–28831 system, where a number of new factors have been identified Bruck I, Kaplan DL (2011a) GINS and Sld3 compete with one another for over the last few years. Many factors involved in CMG Mcm2-7 and Cdc45 binding. J Biol Chem 286:14157–14167 Chromosoma

Bruck I, Kaplan DL (2011b) Origin single-stranded DNA releases Sld3 Evrin C et al (2013) In the absence of ATPase activity, pre-RC formation protein from the Mcm2-7 complex, allowing the GINS tetramer to is blocked prior to MCM2-7 hexamer dimerization. Nucleic Acids bind the Mcm2-7 complex. J Biol Chem 286:18602–18613 Res 41:3162–3172 Bruck I, Kaplan DL (2013) Cdc45 protein-single-stranded DNA interac- Evrin C et al (2014) The ORC/Cdc6/MCM2-7 complex facilitates tion is important for stalling the helicase during replication stress. J MCM2-7 dimerization during prereplicative complex formation. Biol Chem 288:7550–7563 Nucleic Acids Res 42:2257–2269 Brummer A, Salazar C, Zinzalla V, Alberghina L, Hofer T (2010) Fernandez-Cid A et al (2013) An ORC/Cdc6/MCM2-7 complex is Mathematical modelling of DNA replication reveals a trade-off formed in a multistep reaction to serve as a platform for MCM between coherence of origin activation and robustness against double-hexamer assembly. Mol Cell 50:577–588 rereplication. PLoS Comput Biol 6:e1000783. doi:10.1371/journal. Ferreira MF, Santocanale C, Drury LS, Diffley JF (2000) Dbf4p, pcbi.1000783 an essential S phase-promoting factor, is targeted for degra- Charych DH et al (2008) Inhibition of Cdc7/Dbf4 kinase activity affects dation by the anaphase-promoting complex. Mol Cell Biol specific phosphorylation sites on MCM2 in cancer cells. J Cell 20:242–248 Biochem 104:1075–1086 Francis LI, Randell JC, Takara TJ, Uchima L, Bell SP (2009) Chen S, Bell SP (2011) CDK prevents Mcm2-7 helicase loading by Incorporation into the prereplicative complex activates the Mcm2- inhibiting Cdt1 interaction with Orc6. Genes Dev 25:363–372. 7 helicase for Cdc7-Dbf4 phosphorylation. Genes Dev 23:643–654 doi:10.1101/gad.2011511 Frigola J, Remus D, Mehanna A, Diffley JF (2013) ATPase-dependent Chen S, de Vries MA, Bell SP (2007) Orc6 is required for dynamic quality control of DNA replication origin licensing. Nature 495: recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev 339–343. doi:10.1038/nature11920 21:2897–2907 Froelich CA, Kang S, Epling LB, Bell SP, Enemark EJ (2014) A con- Cho WH, Lee YJ, Kong SI, Hurwitz J, Lee JK (2006) CDC7 kinase served MCM single-stranded DNA binding element is essential for phosphorylates serine residues adjacent to acidic amino acids in the replication initiation. Elife 3:e01993. doi:10.7554/eLife.01993 minichromosome maintenance 2 protein. Proc Natl Acad Sci U S A Fu YV et al (2011) Selective bypass of a lagging strand roadblock by the 103:11521–11526 eukaryotic replicative DNA helicase. Cell 146:931–941. doi:10. Chowdhury A et al (2010) The DNA unwinding element binding protein 1016/j.cell.2011.07.045 DUE-B interacts with Cdc45 in preinitiation complex formation. Fukuura M, Nagao K, Obuse C, Takahashi TS, Nakagawa T, Masukata H MolCellBiol30:1495–1507 (2011) CDK promotes interactions of Sld3 and Drc1 with Cut5 for Coleman TR, Carpenter PB, Dunphy WG (1996) The Xenopus Cdc6 initiation of DNA replication in fission yeast. Mol Biol Cell 22: protein is essential for the initiation of a single round of DNA 2620–2633 replication in cell-free extracts. Cell 87:53–63 Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Costa A, Ilves I, Tamberg N, Petojevic T, Nogales E, Botchan MR, Edmondson RD, Labib K (2006) GINS maintains association of Berger JM (2011) The structural basis for MCM2-7 helicase activa- Cdc45 with MCM in replisome progression complexes at eukaryotic tion by GINS and Cdc45. Nat Struct Mol Biol 18:471–477. doi:10. DNA replication forks. Nat Cell Biol 8:358–366 1038/nsmb.2004 Gambus A et al (2009) A key role for Ctf4 in coupling the MCM2-7 Costa A et al (2014) DNA binding polarity, dimerization, and ATPase helicase to DNA polymerase alpha within the eukaryotic replisome. ring remodeling in the CMG helicase of the eukaryotic replisome. Embo J 28:2992–3004 Elife 3:e03273. doi:10.7554/eLife.03273 Gambus A, Khoudoli GA, Jones RC, Blow JJ (2011) MCM2-7 form Dave A, Cooley C, Garg M, Bianchi A (2014) Protein phosphatase 1 double hexamers at licensed origins in Xenopus egg extract. J Biol recruitment by Rif1 regulates DNA replication origin firing by Chem 286:11855–11864 counteracting DDK activity. Cell Rep 7:53–61. doi:10.1016/j. Garcia V, Furuya K, Carr AM (2005) Identification and functional anal- celrep.2014.02.019 ysis of TopBP1 and its homologs. DNA Repair (Amst) 4:1227– Di Perna R, Aria V, De Falco M, Sannino V, Okorokov AL, Pisani FM, 1239 De Felice M (2013) The physical interaction of Mcm10 with Cdc45 Hardy CF, Dryga O, Seematter S, Pahl PM, Sclafani RA (1997) mcm5/ modulates their DNA-binding properties. Biochem J 454:333–343 cdc46-bob1 bypasses the requirement for the S phase activator Diffley JF (2010) The many faces of redundancy in DNA replication Cdc7p. Proc Natl Acad Sci U S A 94:3151–3155 control. Cold Spring Harb Symp Quant Biol 75:135–142. doi:10. Hashimoto Y, Takisawa H (2003) Xenopus Cut5 is essential for a CDK- 1101/sqb.2010.75.062 dependent process in the initiation of DNA replication. Embo J 22: Donovan S, Harwood J, Drury LS, Diffley JF (1997) Cdc6p-dependent 2526–2535 loading of Mcm proteins onto pre-replicative chromatin in budding Hashimoto Y, Puddu F, Costanzo V (2012) RAD51- and MRE11- yeast. Proc Natl Acad Sci U S A 94:5611–5616 dependent reassembly of uncoupled CMG helicase complex at Drury LS, Perkins G, Diffley JF (1997) The Cdc4/34/53 pathway targets collapsed replication forks. Nat Struct Mol Biol 19:17–24 Cdc6p for proteolysis in budding yeast. Embo J 16:5966–5976 Hayano M, Kanoh Y, Matsumoto S, Renard-Guillet C, Shirahige K, Duch A, Palou G, Jonsson ZO, Palou R, Calvo E, Wohlschlegel J, Masai H (2012) Rif1 is a global regulator of timing of replication Quintana DG (2011) A Dbf4 mutant contributes to bypassing the origin firing in fission yeast. Genes Dev 26:137–150 Rad53-mediated block of origins of replication in response to Heller RC, Kang S, Lam WM, Chen S, Chan CS, Bell SP (2011) genotoxic stress. J Biol Chem 286:2486–2491 Eukaryotic origin-dependent DNA replication in vitro reveals se- Edwards AM, Kus B, Jansen R, Greenbaum D, Greenblatt J, quential action of DDK and S-CDK kinases. Cell 146:80–91. doi: Gerstein M (2002) Bridging structural biology and genomics: 10.1016/j.cell.2011.06.012 assessing protein interaction data with known complexes. Hiraga S et al (2014) Rif1 controls DNA replication by directing protein Trends Genet 18:529–536 phosphatase 1 to reverse Cdc7-mediated phosphorylation of the Elsasser S, Chi Y, Yang P, Campbell JL (1999) Phosphorylation controls MCM complex. Genes Dev 28:372–383 timing of Cdc6p destruction: a biochemical analysis. Mol Biol Cell Humphrey T, Pearce A (2005) Cell cycle molecules and mechanisms of 10:3263–3277 the budding and fission yeasts. Methods Mol Biol 296:3–29 Evrin C et al (2009) A double-hexameric MCM2-7 complex is loaded Huo L et al (2012) The Rix1 (Ipi1p-2p-3p) complex is a critical determi- onto origin DNA during licensing of eukaryotic DNA replication. nant of DNA replication licensing independent of their roles in Proc Natl Acad Sci U S A 106:20240–20245 ribosome biogenesis. Cell Cycle 11:1325–1339 Chromosoma

Ilves I, Petojevic T, Pesavento JJ, Botchan MR (2010) Activation of the Liachko I, Tye BK (2009) Mcm10 mediates the interaction between DNA MCM2-7 helicase by association with Cdc45 and GINS proteins. replication and silencing machineries. Genetics 181:379–391 Mol Cell 37:247–258 Liang C, Stillman B (1997) Persistent initiation of DNA replication and Im JS, Ki SH, Farina A, Jung DS, Hurwitz J, Lee JK (2009) Assembly of chromatin-bound MCM proteins during the cell cycle in cdc6 mu- the Cdc45-Mcm2-7-GINS complex in human cells requires the tants. Genes Dev 11:3375–3386 Ctf4/And-1, RecQL4, and Mcm10 proteins. Proc Natl Acad Sci U Liku ME, Nguyen VQ, Rosales AW, Irie K, Li JJ (2005) CDK phosphor- S A 106:15628–15632 ylation of a novel NLS-NES module distributed between two sub- Ishimi Y (1997) A DNA helicase activity is associated with an MCM4, units of the Mcm2-7 complex prevents chromosomal rereplication. −6, and −7 protein complex. J Biol Chem 272:24508–24513 MolBiolCell16:5026–5039 Itou H, Muramatsu S, Shirakihara Y, Araki H (2014) Crystal structure of Lopez-Mosqueda J, Maas NL, Jonsson ZO, Defazio-Eli LG, the homology domain of the eukaryotic DNA replication proteins Wohlschlegel J, Toczyski DP (2010) Damage-induced phosphory- sld3/treslin. Structure 22:1341–1347 lation of Sld3 is important to block late origin firing. Nature 467: Jaspersen SL, Charles JF, Morgan DO (1999) Inhibitory phosphorylation 479–483 of the APC regulator Hct1 is controlled by the kinase Cdc28 and the Makiniemi M et al (2001) BRCT domain-containing protein TopBP1 phosphatase Cdc14. Curr Biol 9:227–236 functions in DNA replication and damage response. J Biol Chem Kamimura Y, Masumoto H, Sugino A, Araki H (1998) Sld2, which 276:30399–30406 interacts with Dpb11 in Saccharomyces cerevisiae, is required for Masai H, Matsui E, You Z, Ishimi Y, Tamai K, Arai K (2000) Human chromosomal DNA replication. Mol Cell Biol 18:6102–6109 Cdc7-related kinase complex. In vitro phosphorylation of MCM by Kamimura Y, Tak YS, Sugino A, Araki H (2001) Sld3, which interacts concerted actions of Cdks and Cdc7 and that of a criticial threonine with Cdc45 (Sld4), functions for chromosomal DNA replication in residue of Cdc7 bY Cdks. J Biol Chem 275:29042–29052 Saccharomyces cerevisiae. Embo J 20:2097–2107 Masai H et al (2006) Phosphorylation of MCM4 by Cdc7 kinase facili- Kanemaki M, Labib K (2006) Distinct roles for Sld3 and GINS during tates its interaction with Cdc45 on the chromatin. J Biol Chem 281: establishment and progression of eukaryotic DNA replication forks. 39249–39261 Embo J 25:1753–1763 Masumoto H, Muramatsu S, Kamimura Y, Araki H (2002) S-Cdk- Kang YH, Farina A, Bermudez VP, Tappin I, Du F, Galal WC, Hurwitz J dependent phosphorylation of Sld2 essential for chromosomal (2013) Interaction between human Ctf4 and the Cdc45/Mcm2-7/ DNA replication in budding yeast. Nature 415:651–655 GINS (CMG) replicative helicase. Proc Natl Acad Sci U S A 110: Matsumoto S, Hayano M, Kanoh Y, Masai H (2011) Multiple pathways 19760–19765 can bypass the essential role of fission yeast Hsk1 kinase in DNA Kanke M, Kodama Y, Takahashi TS, Nakagawa T, Masukata H (2012) replication initiation. J Cell Biol 195:387–401 Mcm10 plays an essential role in origin DNA unwinding after Matsuno K, Kumano M, Kubota Y,Hashimoto Y,Takisawa H (2006) The loading of the CMG components. Embo J 31:2182–2194 N-terminal noncatalytic region of Xenopus RecQ4 is required for Kanter DM, Kaplan DL (2011) Sld2 binds to origin single-stranded DNA chromatin binding of DNA polymerase alpha in the initiation of and stimulates DNA annealing. Nucleic Acids Res 39:2580–2592 DNA replication. Mol Cell Biol 26:4843–4852 Katou Y, Kaneshiro K, Aburatani H, Shirahige K (2006) Genomic Mattarocci S et al (2014) Rif1 controls DNA replication timing in yeast approach for the understanding of dynamic aspect of chromosome through the PP1 phosphatase Glc7. Cell Rep 7:62–69. doi:10.1016/ behavior. Methods Enzymol 409:389–410. doi:10.1016/S0076- j.celrep.2014.03.010 6879(05)09023-3 McIntosh D, Blow JJ (2012) Dormant origins, the licensing checkpoint, Krastanova I, Sannino V, Amenitsch H, Gileadi O, Pisani FM, Onesti S and the response to replicative stresses Cold Spring Harb Perspect (2012) Structural and functional insights into the DNA replication Biol 4. doi:10.1101/cshperspect.a012955 factor Cdc45 reveal an evolutionary relationship to the DHH family Merchant AM, Kawasaki Y, Chen Y, Lei M, Tye BK (1997) A lesion in of phosphoesterases. J Biol Chem 287:4121–4128 the DNA replication initiation factor Mcm10 induces pausing of Kumagai A, Shevchenko A, Dunphy WG (2010) Treslin collaborates elongation forks through chromosomal replication origins in with TopBP1 in triggering the initiation of DNA replication. Cell Saccharomyces cerevisiae. Mol Cell Biol 17:3261–3271 140:349–359 Montagnoli A et al (2006) Identification of Mcm2 phosphorylation sites Kumagai A, Shevchenko A, Dunphy WG (2011) Direct regulation of by S-phase-regulating kinases. J Biol Chem 281:10281–10290 Treslin by cyclin-dependent kinase is essential for the onset of DNA Moyer SE, Lewis PW, Botchan MR (2006) Isolation of the Cdc45/ replication. J Cell Biol 193:995–1007. doi:10.1083/jcb.201102003 Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic Kunkel TA, Burgers PM (2008) Dividing the workload at a eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 103: replication fork Trends. Cell Biol 18:521–527. doi:10.1016/j.tcb. 10236–10241 2008.08.005 Muramatsu S, Hirai K, Tak YS, Kamimura Y, Araki H (2010) CDK- Labib K (2010) How do Cdc7 and cyclin-dependent kinases trigger the dependent complex formation between replication proteins Dpb11, initiation of chromosome replication in eukaryotic cells? Genes Dev Sld2, Pol (epsilon}, and GINS in budding yeast. Genes Dev 24:602–612 24:1208–1219. doi:10.1101/gad.1933010 Nakajima R, Masukata H (2002) SpSld3 is required for loading and Labib K, Diffley JF, Kearsey SE (1999) G1-phase and B-type cyclins maintenance of SpCdc45 on chromatin in DNA replication in fission exclude the DNA-replication factor Mcm4 from the nucleus. Nat yeast. Mol Biol Cell 13:1462–1472 Cell Biol 1:415–422 Natsume T et al (2013) Kinetochores coordinate pericentromeric cohe- Laskey RA, Madine MA (2003) A rotary pumping model for helicase sion and early DNA replication by Cdc7-Dbf4 kinase recruitment. function of MCM proteins at a distance from replication forks. Mol Cell 50:661–674 EMBO Rep 4:26–30. doi:10.1038/sj.embor.embor706 Nguyen VQ, Co C, Li JJ (2001) Cyclin-dependent kinases prevent DNA Lee C, Liachko I, Bouten R, Kelman Z, Tye BK (2010) Alternative re-replication through multiple mechanisms. Nature 411:1068– mechanisms for coordinating polymerase alpha and MCM helicase. 1073. doi:10.1038/35082600 MolCellBiol30:423–435. doi:10.1128/MCB.01240-09 Noguchi E, Shanahan P, Noguchi C, Russell P (2002) CDK phosphory- Lee JK, Hurwitz J (2000) Isolation and characterization of various com- lation of Drc1 regulates DNA replication in fission yeast. Curr Biol plexes of the minichromosome maintenance proteins of 12:599–605 Schizosaccharomyces pombe. J Biol Chem 275:18871–18878. On KF, Beuron F, Frith D, Snijders AP, Morris EP, Diffley JF (2014) doi:10.1074/jbc.M001118200 Prereplicative complexes assembled in vitro support origin- Chromosoma

dependent and independent DNA replication. Embo J 33:605–620. Speck C, Messer W (2001) Mechanism of origin unwinding: sequential doi:10.1002/embj.201387369 binding of DnaA to double- and single-stranded DNA. Embo J 20: Oshiro G, Owens JC, Shellman Y, Sclafani RA, Li JJ (1999) Cell cycle 1469–1476. doi:10.1093/emboj/20.6.1469 control of Cdc7p kinase activity through regulation of Dbf4p stabil- Speck C, Chen Z, Li H, Stillman B (2005) ATPase-dependent cooperative ity.MolCellBiol19:4888–4896 binding of ORC and Cdc6 to origin DNA. Nat Struct Mol Biol 12: Perkins G, Drury LS, Diffley JF (2001) Separate SCF(CDC4) recognition 965–971 elements target Cdc6 for proteolysis in S phase and mitosis. Embo J Sun J et al (2013) Cryo-EM structure of a helicase loading intermediate 20:4836–4845 containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nat Struct Peters JM (2006) The anaphase promoting complex/cyclosome: a ma- MolBiol20:944–951 chine designed to destroy. Nat Rev Mol Cell Biol 7:644–656. doi: Sun J et al (2014) Structural and mechanistic insights into Mcm2-7 10.1038/nrm1988 double-hexamer assembly and function. Genes Dev 28: in press Ramer MD, Suman ES, Richter H, Stanger K, Spranger M, Bieberstein Szambowska A, Tessmer I, Kursula P, Usskilat C, Prus P, Pospiech H, N, Duncker BP (2013) Dbf4 and Cdc7 proteins promote DNA Grosse F (2014) DNA binding properties of human Cdc45 suggest a replication through interactions with distinct Mcm2-7 protein sub- function as molecular wedge for DNA unwinding. Nucleic Acids units. J Biol Chem 288:14926–14935 Res 42:2308–2319 Randell JC, Bowers JL, Rodriguez HK, Bell SP (2006) Sequential ATP Tak YS, Tanaka Y, Endo S, Kamimura Y, Araki H (2006) A CDK- hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 catalysed regulatory phosphorylation for formation of the DNA helicase. Mol Cell 21:29–39 replication complex Sld2-Dpb11. Embo J 25:1987–1996. doi:10. Randell JC, Fan A, Chan C, Francis LI, Heller RC, Galani K, Bell SP 1038/sj.emboj.7601075 (2010) Mec1 is one of multiple kinases that prime the Mcm2-7 Takara TJ, Bell SP (2011) Multiple Cdt1 molecules act at each origin to helicase for phosphorylation by Cdc7. Mol Cell 40:353–363. doi: load replication-competent Mcm2-7 helicases. Embo J 30:4885– 10.1016/j.molcel.2010.10.017 4896 Rappas M, Oliver AW, Pearl LH (2011) Structure and function of Takayama Y, Kamimura Y, Okawa M, Muramatsu S, Sugino A, Araki H the Rad9-binding region of the DNA-damage checkpoint (2003) GINS, a novel multiprotein complex required for chromo- adaptor TopBP1. Nucleic Acids Res 39:313–324. doi:10. somal DNA replication in budding yeast. Genes Dev 17:1153–1165 1093/nar/gkq743 Tanaka H et al (2009) Ctf4 coordinates the progression of helicase and Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF (2009) DNA polymerase alpha. Genes Cells 14:807–820 Concerted loading of Mcm2-7 double hexamers around DNA dur- Tanaka S, Araki H (2013) Helicase activation and establishment of repli- ing DNA replication origin licensing. Cell 139:719–730 cation forks at chromosomal origins of replication. Cold Spring Harb Renard-Guillet C, Kanoh Y, Shirahige K, Masai H (2014) Temporal and Perspect Biol 5:a010371. doi:10.1101/cshperspect.a010371 spatial regulation of eukaryotic DNA replication: from regulated Tanaka S, Diffley JF (2002) Interdependent nuclear accumulation of initiation to genome-scale timing program. Semin Cell Dev Biol budding yeast Cdt1 and Mcm2-7 during G1 phase. Nat Cell Biol 30C:110–120. doi:10.1016/j.semcdb.2014.04.014 4:198–207 Riera A, Tognetti S, Speck C (2014) Helicase loading: how to build a Tanaka T, Knapp D, Nasmyth K (1997) Loading of an Mcm protein onto MCM2-7 double-hexamer. Semin Cell Dev Biol 30C:104–109 DNA replication origins is regulated by Cdc6p and CDKs. Cell 90: Saka Y, Fantes P, Sutani T, McInerny C, Creanor J, Yanagida M (1994) 649–660 Fission yeast cut5 links nuclear chromatin and M phase regulator in Tanaka S, Umemori T, Hirai K, Muramatsu S, Kamimura Y, Araki H the replication checkpoint control. Embo J 13:5319–5329 (2007) CDK-dependent phosphorylation of Sld2 and Sld3 initiates Samel SA et al (2014) A unique DNA entry gate serves for regulated DNA replication in budding yeast. Nature 445:328–332 loading of the eukaryotic replicative helicase MCM2–7ontoDNA. Tanaka S, Nakato R, Katou Y, Shirahige K, Araki H (2011) Origin Genes Dev 28:1653–1666 association of Sld3, Sld7, and Cdc45 proteins is a key step for Sanchez-Pulido L, Ponting CP (2011) Cdc45: the missing RecJ ortholog determination of origin-firing timing. Curr Biol 21:2055–2063 in eukaryotes? Bioinformatics 27:1885–1888. doi:10.1093/ Tanaka S, Komeda Y, Umemori T, Kubota Y, Takisawa H, Araki H bioinformatics/btr332 (2013) Efficient initiation of DNA replication in eukaryotes requires Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG, Dpb11/TopBP1-GINS interaction. Mol Cell Biol 33:2614–2622 Venkitaraman AR (2005) Initiation of DNA replication requires the van Deursen F, Sengupta S, De Piccoli G, Sanchez-Diaz A, Labib K RECQL4 protein mutated in Rothmund-Thomson syndrome. Cell (2012) Mcm10 associates with the loaded DNA helicase at replica- 121:887–898 tion origins and defines a novel step in its activation. Embo J 31: Schmidt U, Wollmann Y, Franke C, Grosse F, Saluz HP, Hanel F (2008) 2195–2206 Characterization of the interaction between the human DNA topo- Van Hatten RA, Tutter AV, Holway AH, Khederian AM, Walter JC, isomerase IIbeta-binding protein 1 (TopBP1) and the cell division Michael WM (2002) The Xenopus Xmus101 protein is required cycle 45 (Cdc45) protein. Biochem J 409:169–177 for the recruitment of Cdc45 to origins of DNA replication. J Cell Sheu YJ, Stillman B (2006) Cdc7-Dbf4 phosphorylates MCM proteins Biol 159:541–547 via a docking site-mediated mechanism to promote S phase progres- Wang G, Tong X, Weng S, Zhou H (2012) Multiple phosphorylation of sion. Mol Cell 24:101–113 Rad9 by CDK is required for DNA damage checkpoint activation. Sheu YJ, Stillman B (2010) The Dbf4-Cdc7 kinase promotes S phase by Cell Cycle 11:3792–3800. doi:10.4161/cc.21987 alleviating an inhibitory activity in Mcm4. Nature 463:113–117. doi: Warren EM, Vaithiyalingam S, Haworth J, Greer B, Bielinsky AK, 10.1038/nature08647 Chazin WJ, Eichman BF (2008) Structural basis for DNA binding Siddiqui K, On KF, Diffley JF (2013) Regulating DNA replication in eukarya by replication initiator Mcm10. Structure 16:1892–1901. doi:10. Cold Spring Harb Perspect Biol 5. doi:10.1101/cshperspect.a012930 1016/j.str.2008.10.005 Simon AC et al (2014) A Ctf4 trimer couples the CMG helicase to DNA Watase G, Takisawa H, Kanemaki MT (2012) Mcm10 plays a role in polymerase alpha in the eukaryotic replisome. Nature. doi:10.1038/ functioning of the eukaryotic replicative DNA helicase, Cdc45- nature13234 Mcm-GINS. Curr Biol 22:343–349 Skarstad K, Katayama T (2013) Regulating DNA replication in bacteria. Weinreich M, Stillman B (1999) Cdc7p-Dbf4p kinase binds to chromatin Cold Spring Harb Perspect Biol 5:a012922. doi:10.1101/ during S phase and is regulated by both the APC and the RAD53 cshperspect.a012922 checkpoint pathway. Embo J 18:5334–5346 Chromosoma

Weinreich M, Liang C, Stillman B (1999) The Cdc6p nucleotide-binding Yardimci H et al (2012) Bypass of a protein barrier by a replicative DNA motif is required for loading Mcm proteins onto chromatin. Proc helicase. Nature 492:205–209. doi:10.1038/nature11730 Natl Acad Sci U S A 96:441–446 Zachariae W, Schwab M, Nasmyth K, Seufert W (1998) Control of cyclin Wohlschlegel JA, Dhar SK, Prokhorova TA, Dutta A, Walter JC (2002) ubiquitination by CDK-regulated binding of Hct1 to the anaphase Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 promoting complex. Science 282:1721–1724 and stimulates origin binding of Cdc45. Mol Cell 9:233–240 Zegerman P, Diffley JF (2007) Phosphorylation of Sld2 and Sld3 by Xu X, Rochette PJ, Feyissa EA, Su TV, Liu Y (2009) MCM10 mediates cyclin-dependent kinases promotes DNA replication in budding RECQ4 association with MCM2-7 helicase complex during DNA yeast. Nature 445:281–285 replication. Embo J 28:3005–3014 Zegerman P, Diffley JF (2010) Checkpoint-dependent inhibition of DNA Yabuuchi H, Yamada Y, Uchida T, Sunathvanichkul T, Nakagawa T, replication initiation by Sld3 and Dbf4 phosphorylation. Nature 467: Masukata H (2006) Ordered assembly of Sld3, GINS and Cdc45 474–478 is distinctly regulated by DDK and CDK for activation of replication Zhou Y, Wang TS (2004) A coordinated temporal interplay of origins. Embo J 25:4663–4674 nucleosome reorganization factor, sister chromatin cohesion Yamada Y, Nakagawa T, Masukata H (2004) A novel intermediate in factor, and DNA polymerase alpha facilitates DNA replica- initiation complex assembly for fission yeast DNA replication. Mol tion.MolCellBiol24:9568–9579. doi:10.1128/MCB.24.21. Biol Cell 15:3740–3750. doi:10.1091/mbc.E04-04-0292 9568-9579.2004 Yardimci H, Loveland AB, Habuchi S, van Oijen AM, Walter JC (2010) Zou L, Stillman B (1998) Formation of a preinitiation complex by S- Uncoupling of sister replisomes during eukaryotic DNA replication. phase cyclin CDK-dependent loading of Cdc45p onto chromatin. Mol Cell 40:834–840. doi:10.1016/j.molcel.2010.11.027 Science 280:593–596