Cellular replicative DNA helicases are hexameric ring-shaped motors that track on single-strand (ss) DNA, acting as a wedge to unwind the duplex (Lyubimov et al., 2011). In bacteria, the circular helicase is a homohexamer that directly assembles onto an AT-rich ssDNA region melted by initiator proteins, but in eukaryotes the origin recognition complex (ORC) does not unwind DNA (Bleichert et al., 2017). Instead, in G1-phase, two Mcm2-7 hexamer rings are assembled head-to-head (N-to-N) around double-strand (ds) DNA by ORC, Cdt1 and Cdc6 (Bell and Labib, 2016; Evrin et al., 2009; O'Donnell et al., 2013; Remus et al., 2009). In S-phase, additional factors assemble Cdc45 and GINS onto each Mcm2-7 to form two replicative helicases on dsDNA oriented head-to-head (Ilves et al., 2010; Moyer et al., 2006; Parker et al., 2017). Each helicase contains 11 different subunits, and the term CMG (Cdc45, Mcm2-7, GINS) was coined because it contains Cdc45, Mcm2-7, and the four subunit GINS complex (Ilves et al., 2010; Moyer et al., 2006; Parker et al., 2017). Mcm10 is required to activate each CMG to encircle ssDNA for bidirectional replication (Douglas et al., 2018; Heller et al., 2011; Kanke et al., 2012; van Deursen et al., 2012; Watase et al., 2012; Yeeles et al., 2015). However, the detailed processes of origin melting and the transition of CMG from dsDNA to ssDNA translocation are unknown. In particular, it is not clear whether CMG and Mcm10 alone are sufficient to mediate this transition or whether other factors required for CMG assembly are also involved.

Recent electron microscopy (EM) studies of Saccharomyces cerevisiae (S.c.) CMG at a replication fork show that CMG tracks on ssDNA with its N-tier facing the fork (Douglas et al., 2018; Georgescu et al., 2017). N-first movement of CMG implies that head-to-head CMGs at the origin are directed toward one another when assembled around duplex DNA. Thus, each CMG blocks the forward movement of the other, and the duplex must be unwound a sufficient distance for both CMGs to expel one strand for their transition from dsDNA to ssDNA, which they must do to pass one another and form bidirectional forks (Figure 1). These last steps of origin initiation require the Mcm10 protein (Douglas et al., 2018; Kanke et al., 2012; van Deursen et al., 2012; Watase et al., 2012; Yeeles et al., 2015). Mcm10 contains a DNA binding region (Warren et al., 2008) and interaction with the N-terminal regions of Mcm2 and Mcm6 has been demonstrated (Douglas and Diffley, 2016; Lõoke et al., 2017). Recent cross-linking/mass spectrometry studies reveal a large surface area of CMG-Mcm10 interaction that includes 6 of the 11 CMG subunits, mostly at the N-surface of CMG but also many sites at the edges and C-surface of CMG (Mayle et al., 2019).

Figure 1

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The mechanism of CMG + Mcm10 (CMGM) function in the final step of origin initiation has been difficult to probe experimentally. Recent studies of CMG formation at origins demonstrate a disruption of tight connections between the Mcm2-7 hexamers upon maturation to CMGs and untwisting of ~0.7 turns of the double helix per CMG (Douglas et al., 2018). However, even if the observed untwisting was due to actual DNA melting, it is too little for CMG to transition to ssDNA (Douglas et al., 2018). Thus, the mechanism of how CMG opens the duplex a sufficient length to transition to ssDNA and the role of Mcm10 in this process remain open questions. In this report we use recombinant CMG and Mcm10 and find that, together, they produce sufficient force to open several turns of the duplex while encircling dsDNA, a sufficient length that would enable CMGs to transition onto ssDNA. Hence, initial unwinding of DNA by CMG-Mcm10 can occur without the participation of other replication initiation factors.

To investigate the activity of the CMG motor while encircling dsDNA, we designed three T-shaped DNA substrates that block passive sliding over the two non-homologous arms at the T-junction (Figure 2A and Figure 2—figure supplement 1). If CMG is not passive, but instead CMG motors on dsDNA with force, it may melt the T-junction arms and produce unwound products, provided the arms are not too long for CMG to melt. Similar substrates have been used to study ring-shaped helicases that track on dsDNA with force including E. coli DnaB helicase and the FtsK chromosome segregation motor (Bigot et al., 2005; Crozat and Grainge, 2010; Kaplan and O'Donnell, 2004; Lyubimov et al., 2011). The T-substrates contain a 3’ ssDNA dT tail that is required for CMG loading (Figure 2a and Figure 2—figure supplement 2) followed by a flush ss/dsDNA junction that enables CMG to move onto dsDNA without unwinding it (Kang et al., 2012; Langston and O'Donnell, 2017) after which it encounters the T-junction block of non-homologous arms (Figure 2a). The three T-shaped DNAs are identical except for the length of the branched arms (20, 30 or 60 bp). The non-homologous arms are annealed to a ³²P-radiolabeled ‘C’ oligo. Proceeding through the T-arm blocks requires that CMG translocates with force while surrounding dsDNA to simultaneously melt both arms, so a T-substrate with 30 bp arms requires melting a total of 60 bp to release the ³²P-C oligo.

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CMG was pre-incubated with the T-substrates for 10’ and then ATP was added to initiate unwinding as illustrated in Figure 2A. An excess of unlabeled C oligo was added 1’ later as a trap to prevent re-annealing of any unwound ³²P-C oligo. CMG has only weak activity in melting the 20 bp arms and cannot destabilize the 30 bp or 60 bp arms (Figure 2B,D). However, upon adding Mcm10, CMG unwound all three T-substrates (Figure 2C,D). Mcm10 alone has no activity in these assays (Figure 2—figure supplement 3). The results are consistent with prior studies showing Mcm10 stimulates CMG helicase activity, enhances CMG processivity during unwinding (Langston et al., 2017; Lõoke et al., 2017), and is required to activate CMG unwinding at origins (Douglas et al., 2018; Kanke et al., 2012; van Deursen et al., 2012; Watase et al., 2012; Yeeles et al., 2015). Possible roles of Mcm10 in promoting CMG unwinding while encircling dsDNA are presented in the Discussion. The extent of unwinding decreases as the length of the non-homologous arms increases, suggesting either limited processivity of CMG + Mcm10 in directional dsDNA translocation or that CMG expels the non-tracking strand and transitions to ssDNA translocation while unwinding the longer T-substrates; strand expulsion would result in only partial unwinding followed by rapid intramolecular reannealing to reform the starting substrate, a topic we examine later.

Previous studies of multi-subunit, ring-shaped motors that surround and track on dsDNA show that they primarily contact one strand of the duplex. Two of the most well-studied examples are the phi29 DNA packaging motor, which tracks on only one strand while encircling dsDNA (Aathavan et al., 2009) and the bacterial clamp loader that also surrounds dsDNA but contacts only one strand (Simonetta et al., 2009) (Figure 3—figure supplement 1). Similarly, the cryoEM structure of CMG on forked DNA shows that the translocation strand adopts a B-form spiral shape in the motor domains (Figure 3—figure supplement 1), implying that the motors need not alter conformation to encircle dsDNA (Abid Ali et al., 2016; Georgescu et al., 2017). A corresponding spiral of ssDNA in the central channel is a common feature of all replicative helicase structures examined thus far (Gao et al., 2019; O'Donnell and Li, 2018; Parker et al., 2017). These findings suggest CMG may track on one strand while encircling dsDNA. Translocases bind the negative phosphate backbone for mobility (Kaplan and O'Donnell, 2004; Lyubimov et al., 2011; Parker et al., 2017), and thus to test CMG for tracking on one strand of dsDNA we placed ten neutral methylphosphonate linkages on either strand of the duplex preceding the T-substrate arms (Figure 3A and Figure 3—figure supplement 2), a strategy used to identify the tracking strand for the phi29 motor (Aathavan et al., 2009). When the methylphosphonate linkages were on the 3’−5’ strand in the direction of unwinding, duplex unwinding was significantly reduced compared to methylphosphonate linkages on the opposite strand (Figure 3B,C). This result is consistent with CMG tracking mainly on the 3’−5’ strand while surrounding dsDNA, which is also the tracking strand for helicase activity while encircling ssDNA. This is noteworthy given that in the inactive Mcm2-7 double hexamer, each ring makes an equal number of contacts with the 3'−5' and 5'−3' strands (Abid Ali et al., 2017; Noguchi et al., 2017). Hence, duplex DNA interaction by the MCM and by the CMG is fundamentally different.

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The studies of Figures 2 and 3 show that CMG + Mcm10 tracks on dsDNA with force and interacts with the 3’−5’ strand more than the 5’−3’ strand. On the basis of these observations, we hypothesized that head-to-head CMGs encircling dsDNA and pushing against one another at origins would pull on opposite strands of the duplex, providing the force necessary to melt the origin. To test this, we designed a linear duplex with CMG loading sites at either end to simulate an origin in which two CMGs are oriented head-to-head on dsDNA (Figure 4A). The substrate supports loading of two (at least) oppositely oriented CMG complexes onto duplex DNA that will translocate toward one another and collide in the same way that CMGs do at an origin but without the need for CMG helicase assembly by multiple initiation factors (Figure 4A, middle). This allows us to determine if the duplex melting activity required at origins is inherent in head-to-head CMG + Mcm10 complexes or if additional factors are needed. In this assay, if the substrate is unwound, the trap oligo prevents reannealing by forming a fork with the unwound ³²P-labeled strand and the fork structure migrates more slowly in the gel than the initial substrate (Figure 4A, bottom).

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Considering that circular helicases like CMG are inefficient in threading onto DNA, we assume that binding of two CMG to a single substrate is probably a rare event. To further reduce the probability of >2 CMG loading, we minimized the opportunity for CMG to bind to the substrate as follows. First, CMG was mixed with the substrate on ice and then incubated for 1 min at 30°C in the absence of ATP to allow the mixture to reach the reaction temperature. Next, ATP was added to initiate CMG loading and duplex translocation along with Mcm10 to initiate duplex melting (Douglas et al., 2018; Kanke et al., 2012; van Deursen et al., 2012; Watase et al., 2012; Yeeles et al., 2015). Then 45 s after adding ATP and Mcm10, we added the trap oligo which quenches further CMG loading (Figure 4—figure supplement 1). As shown in Figure 4B (lanes 1–5), CMG readily unwinds the two-tailed substrate in the presence of Mcm10. Moreover, unwinding continues well beyond the time of addition of the trap, thereby excluding the possibility that additional CMG loading events are required for unwinding (see Figure 4B and C; also see Figure 4—figure supplement 1 for effectiveness of the trap). The reaction is absolutely dependent on addition of Mcm10 (Figure 4—figure supplement 2) and also requires ATP and CMG (Figure 4—figure supplement 3), showing that the observed unwinding is the product of CMG’s ATP-dependent motor activity coupled to Mcm10 function rather than the product of either CMG or Mcm10 acting separately. The reaction also requires loading of head-to-head CMG complexes because duplexes with only a single CMG loading tail at one end or the other were essentially inactive compared to the two-tailed substrate (Figure 4B, lanes 6–15 and Figure 4C). In overview, these results demonstrate that head-to-head CMG complexes, along with Mcm10, can perform the DNA melting required at origins of replication without the participation of any other factors. Furthermore, the results are consistent with the action of two independent CMGs colliding with one another on dsDNA, in contrast to the highly interconnected and interdependent but inactive Mcm2-7 complexes within the double hexamer.

Two explanations for unwinding by head-to-head CMG-Mcm10s are illustrated in Figure 4A. One process involves strand shearing (Figure 4A, left arrow) as observed in the reactions using the T-substrate in which the two non-homologous arms are sheared apart (Figures 2 and 3). An alternative process is that once sufficient dsDNA is unwound, the non-tracking strand may be expelled from the inner chamber of CMG, enabling completion of unwinding by conventional CMG helicase activity in which the CMGs encircle and translocate 3’−5’ on ssDNA (Figure 4A, right arrow).

In an effort to address whether strand expulsion occurs during unwinding, we constructed a modified T-substrate in which the two 50 bp non-complementary arms of the T-substrate are annealed to separate oligos with 5’ flaps, allowing us to monitor unwinding of each arm independently of the other (Figure 5A). This is in contrast to the substrate in Figure 2 where both T-arms had to be melted in order to observe unwinding (see Figure 5—figure supplement 1). For the substrate in Figure 5, the 5’ flap in the ‘C’ oligo on the upper arm of the substrate (Figure 5A, left) was created by using a reverse polarity 3’−3’ linkage near the base of the flap to prevent CMG loading onto what would otherwise be a 3’ flap (Figure 5—figure supplement 2). The other 5’ flapped oligo, on the lower arm, is referred to here as the ‘D’ oligo. For these reactions, two trap oligos are used to prevent unwound products from reannealing: ^trapE prevents unwound oligo C from reannealing to oligo B while ^trapA is an unlabeled version of oligo A that prevents unwound oligos B and/or D from reannealing to radiolabeled oligo A (Figure 5A, right). This experimental design enables visualization of all possible product outcomes of CMGM translocation on dsDNA, including whether CMG continually encircles dsDNA or whether it might expel one strand to encircle only the tracking strand at some point in the process (see Figure 5A,D).

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When CMG loads onto the initial duplex segment of the modified substrate it can follow one of two pathways. If CMG translocates exclusively on duplex DNA during the reaction and melts both non-homologous arms, then both flapped oligos (oligos C and D in Figure 5A, middle right) will be released by the action of the CMG motor. Alternatively, it is possible that CMG will melt only oligo D (Figure 5A, top right). For example, if CMG initially translocates on dsDNA and melts a portion of the non-homologous arms and then expels the non-tracking strand, only the lower flapped oligo D will be unwound (Figure 5A,D). This same outcome could also arise if CMG remains on dsDNA but is faster in melting oligo D compared to oligo C (Figure 5A,E). Alternatively, despite the lack of a 5’ flap on the initial flush duplex segment, CMG might take advantage of thermal fraying at the flush end and unwind the 3’-tailed oligo (³²P-oligo A in Figure 5A, bottom right) from the rest of the substrate. Previous experiments with similar substrates indicate that unwinding of flush duplexes is negligible (Langston and O'Donnell, 2017). By radiolabeling the 3’-tailed ‘A’ oligo, these potential products are observed as separate bands in a native gel (Figure 5B). The reaction conditions are identical to those of the experiments in Figure 2C (CMG + Mcm10) except for the modified substrate design and the use of two traps to prevent unwound substrates from reanealing (see Figure 5A, right).

The results of the experiment with the modified substrate reveal that melting of oligo D is the most prominent product of the reaction, appearing as a clear and distinct band in the gel as early as 90’ after starting the reaction (band labeled *ABC in Figure 5B) and occurring on 40% of substrates over the full time course (Figure 5C). In contrast, the product expected for CMG encircling dsDNA and melting both arms simultaneously was ~10% of total substrate (*AB in Figure 5B,C), as observed in Figure 2 for the 60 bp arm substrate. Continuous ssDNA translocation during the entire experiment was a minor outcome (~5%, *A in Figure 5B,C) as expected because there is no 5’ flap on the initial duplex segment, further supporting the conclusion that CMG moves smoothly from ssDNA onto a flush duplex without unwinding it, as previously observed (Langston and O'Donnell, 2017).

As discussed above, the melting of oligo D may be explained either by CMG +Mcm10 expelling the non-tracking strand (Figure 5D) or by CMG +Mcm10 continuously encircling dsDNA but melting oligo D faster than oligo C (Figure 5E). While we cannot distinguish between these two mechanisms, we note that an ssDNA gate was previously demonstrated for Drosophila CMG (Ilves et al., 2010; Moyer et al., 2006), and subsequently shown to generalize to human CMG and S. cerevisiae CMG (Kang et al., 2012; Langston and O'Donnell, 2017). Also, in recent single molecule studies, we show that CMGMs act as individual particles and that CMGM on dsDNA, upon encountering an RPA-coated fork, expels one strand and transitions to encircle ssDNA where it moves slowly to unwind the duplex (Wasserman et al., 2018). Regardless, the ensemble experiments of Figure 5 cannot rigorously distinguish whether CMG +Mcm10 expels the non-tracking strand or continuously encircles dsDNA to melt oligo D to form the ABC product.

The results presented in this report demonstrate that CMG + Mcm10 translocates on dsDNA with sufficient force to melt at least 60 bp (Figure 2), and that head-to-head CMGs can melt up to 150 bp (Figure 4). The head-to-head CMGs can be added individually and thus are uncoupled and do not need to be intimately connected as in the Mcm2-7 double hexamer. The experiments further demonstrate that Mcm10 is required for these CMG actions and that no other origin firing factors are needed. CMGM tracking on dsDNA with force preferentially interacts with the 3'−5' strand more than the 5'−3' strand, unlike the inactive Mcm2-7 double hexamer that does not unwind DNA and has an equal number of contacts with each strand of duplex DNA (Abid Ali et al., 2017; Noguchi et al., 2017). DNA melting by head-to-head CMGs directed against one another is expected to require a force of 65 pN, the force demonstrated to melt dsDNA in single-molecule experiments with optical traps attached to the ends of opposite strands of a linear duplex (King et al., 2016; van Mameren et al., 2009). Generation of 50–60 pN force has precedent in the ring-shaped FtsK segregation motor (Pease et al., 2005) and in phage packaging motors (Smith et al., 2001). In the case of origin melting, two CMG + Mcm10 are utilized and thus each CMG would only need half the 65 pN required for the ds-to-ssDNA phase transition.

DNA melting at an origin is a necessity for replication in all cell types, but occurs in different ways in bacteria and eukaryotes (reviewed in Lyubimov et al., 2011 and Parker et al., 2017). While bacterial origin binding proteins melt enough DNA for two hexameric helicases to assemble directly onto ssDNA, eukaryotic ORC (Origin Recognition Complex) does not unwind DNA and instead conspires with many other factors to assemble two CMG helicases on dsDNA that are oriented head-to-head (Bell and Labib, 2016). Each CMG is demonstrated to untwist about 0.7 turns apiece (Douglas et al., 2018), but how the CMGs lead to sufficient DNA melting for strand expulsion and pass one another has been unknown except that Mcm10 is required (reviewed in Bell and Labib, 2016).

While the studies of this report provide a mechanistic overview of the origin unwinding step, additional details of this step remain to be determined. Mainly, we do not know the basis by which Mcm10 provides CMG the ability to unwind duplex while encircling duplex DNA. One possibility is that Mcm10 prevents slippage or backtracking by CMG, as backtracking in a ring shaped helicase has been documented previously (Sun et al., 2011). Indeed, recent studies show that the two CMGs at an origin can drift apart in buffer lacking ATP, indicating they are no longer tightly attached as in the Mcm2-7 double hexamer and are capable of backward slippage (Douglas et al., 2018). If Mcm10 were to prevent CMG backtracking it might more efficiently utilize the intrinsic force generated by ATP-driven CMG translocation. We do not know if Mcm10 acts on CMG at origins the same way as it acts upon CMG at replication forks. Unfortunately, CMG-Mcm10 complex with or without a replication fork shows no additional Mcm10 density in cryoEM suggesting Mcm10 flexibility on CMG or multiple conformers of Mcm10 on CMG (Douglas et al., 2018; Mayle et al., 2019), and therefore additional studies will be needed to understand how Mcm10 acts upon CMG. For example, as an alternative to preventing backtracking, Mcm10 may induce a change in DNA structure that makes it easier to melt, as observed in monomeric helicases that orient the ssDNA and dsDNA at nearly right angles (Gao et al., 2019; Lee and Yang, 2006). In practice, the challenge to studying this process is that once two CMGs are formed, Mcm10 binding to the CMGs does not form a stable intermediate to examine because the CMGs quickly pass one another and form replication forks. The structure of CMG-Mcm10 intermediate species at an origin must await further studies. Hence, we propose below a plausible mechanism for Mcm10-dependent origin melting by CMG that utilizes the facts as we know them thus far.

We have shown previously that the N-terminal zinc fingers of CMG encircle and interact with both strands of duplex DNA just prior to the split point and that the duplex proceeds nearly straight into CMG before the leading strand enters the central channel (Georgescu et al., 2017; O'Donnell and Li, 2018). Furthermore, observations of Mcm2-7 head-to-head double hexamers on DNA show they are nearly in-line with one another while they encircle dsDNA (Abid Ali et al., 2017; Li et al., 2015; Noguchi et al., 2017; Remus et al., 2009). Thus we propose a model in Figure 6 for origin melting assuming two in-line head-to-head CMGs (+Mcm10) at an origin. The length of the central channel of each CMG is about 110 Å which can enclose 20–30 bp of dsDNA. Thus, significant melting is required to provide ssDNA of sufficient length for one strand to be excluded from the central channel to the outside of the CMG particle. The motors of CMG are within the C-terminal domains and are separated by the N-terminal domains in a head-to-head orientation (Figure 6). Recent studies show that each CMG can untwist about 7 bp without Mcm10, probably within the C-terminal domains containing the ATP sites, but whether this is unwound DNA or untwisted DNA is not certain (Douglas et al., 2018). While this is insufficient for the dsDNA-to-ssDNA transition, it may be expected to facilitate the transition. Like other ring-shaped oligomers that encircle dsDNA (Aathavan et al., 2009; Simonetta et al., 2009), if CMG tracks primarily on one strand of dsDNA, as indicated by data of Figure 3, the dsDNA will come under tension, each strand being pulled in the opposite direction. Upon reaching the threshold tension for melting, the dsDNA between the CMGs will be sheared to ssDNAs and further tracking will produce additional ssDNA (Figure 6). The disposition of this sheared DNA is not clear, but one possibility is that the ssDNA might be stored within the N-tier by a conserved MCM-ssDNA binding motif, as revealed in a structure of the N-tier of an archaeal MCM bound to ssDNA (Froelich et al., 2014). Alternatively, DNA melting may occur within individual CMG-Mcm10 molecules, leaving DNA between them as duplex. These various possible intermediate states are indicated by the question mark in Figure 6.

Figure 6

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Once sufficient DNA is unwound, the CMGs must expel one strand of ssDNA from the central channel to become bone fide helicases that can pass one another to initiate two divergent replication forks. Expulsion of a ssDNA implies a ssDNA gate within CMG. A ssDNA gate intrinsic to CMG is consistent with the demonstrated ability of CMG to self-load onto circular ssDNA for removal of 5’ flap oligonucleotides bound to the circular DNA, demonstrated for Drosophila CMG and later for human and budding yeast CMG (Ilves et al., 2010; Kang et al., 2012; Langston et al., 2014). Turns in the ssDNA strands that remain after melting the dsDNA would not encumber these transitions because the turns can diffuse into the flanking dsDNA and be removed by topoisomerases (Postow et al., 1999). For example, the two CMG-Mcm10 complexes may rotate in opposite directions relative to one another, following the contour of the DNA (Figure 6), or the DNAs may rotate while the head-to-head CMG-Mcm10’s remain stationary. In either case, the turns in the melted DNA will be pushed out the C-face of each CMG-Mcm10 complex to yield supercoils for topoisomerase action. Once the CMGs pass one another, they have been demonstrated to recruit Pol α-primase to form primed sites that, upon extension, become leading strands (Aria and Yeeles, 2019).

Despite the knowledge gained by the current report, it will require further study to understand the exact details in these last steps in origin initiation in which the CMGs unwind DNA while encircling dsDNA and transition to encircling ssDNA for bidirectional replication. Not only do we require further information on the exact role of Mcm10, but the presumed ssDNA gate has yet to be identified. In fact, many of the preceding steps leading up to assembly of CMG are still not characterized in molecular detail. These include the structural transitions of Mcm2-7 that occur upon DDK phosphorylation that enable it to bind Sld3/7 and Cdc45; the structure of the Mcm2-7-Sld3/7-Cdc45 intermediate; and the structure of the Dbp11-Sld2-Sld3 complex and its interaction with GINS and Pol ε, all of which are required to form CMG from its component parts. Clearly, there is much more to be learned about origin initiation, and given the conservation of the factors involved in higher eukaryotes, the processes discovered in S. cerevisiae will likely generalize.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Aathavan K
   2. Politzer AT
   3. Kaplan A
   4. Moffitt JR
   5. Chemla YR
   6. Grimes S
   7. Jardine PJ
   8. Anderson DL
   9. Bustamante C

   (2009) Substrate interactions and promiscuity in a viral DNA packaging motor

   Nature 461:669–673.

   https://doi.org/10.1038/nature08443

   • PubMed
   • Google Scholar
2. 1. Abid Ali F
   2. Renault L
   3. Gannon J
   4. Gahlon HL
   5. Kotecha A
   6. Zhou JC
   7. Rueda D
   8. Costa A

   (2016) Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

   Nature Communications 7:10708.

   https://doi.org/10.1038/ncomms10708

   • PubMed
   • Google Scholar
3. 1. Abid Ali F
   2. Douglas ME
   3. Locke J
   4. Pye VE
   5. Nans A
   6. Diffley JFX
   7. Costa A

   (2017) Cryo-EM structure of a licensed DNA replication origin

   Nature Communications 8:2241.

   https://doi.org/10.1038/s41467-017-02389-0

   • PubMed
   • Google Scholar
4. 1. Abid Ali F
   2. Costa A

   (2016) The MCM helicase motor of the eukaryotic replisome

   Journal of Molecular Biology 428:1822–1832.

   https://doi.org/10.1016/j.jmb.2016.01.024

   • PubMed
   • Google Scholar
5. 1. Aria V
   2. Yeeles JTP

   (2019) Mechanism of bidirectional Leading-Strand synthesis establishment at eukaryotic DNA replication origins

   Molecular Cell 73:199–211.

   https://doi.org/10.1016/j.molcel.2018.10.019

   • Google Scholar
6. 1. Bell SP
   2. Labib K

   (2016) Chromosome duplication in Saccharomyces cerevisiae

   Genetics 203:1027–1067.

   https://doi.org/10.1534/genetics.115.186452

   • PubMed
   • Google Scholar
7. 1. Bigot S
   2. Saleh OA
   3. Lesterlin C
   4. Pages C
   5. El Karoui M
   6. Dennis C
   7. Grigoriev M
   8. Allemand JF
   9. Barre FX
   10. Cornet F

   (2005) KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase

   The EMBO Journal 24:3770–3780.

   https://doi.org/10.1038/sj.emboj.7600835

   • PubMed
   • Google Scholar
8. 1. Bleichert F
   2. Botchan MR
   3. Berger JM

   (2017) Mechanisms for initiating cellular DNA replication

   Science 355:eaah6317.

   https://doi.org/10.1126/science.aah6317

   • PubMed
   • Google Scholar
9. 1. Costa A
   2. Renault L
   3. Swuec P
   4. Petojevic T
   5. Pesavento JJ
   6. Ilves I
   7. MacLellan-Gibson K
   8. Fleck RA
   9. Botchan MR
   10. Berger JM

   (2014) DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome

   eLife 3:e03273.

   https://doi.org/10.7554/eLife.03273

   • PubMed
   • Google Scholar
10. 1. Crozat E
    2. Grainge I

    (2010) FtsK DNA translocase: the fast motor that knows where it's going

    ChemBioChem 11:2232–2243.

    https://doi.org/10.1002/cbic.201000347

    • PubMed
    • Google Scholar
11. 1. Douglas ME
    2. Ali FA
    3. Costa A
    4. Diffley JFX

    (2018) The mechanism of eukaryotic CMG helicase activation

    Nature 555:265–268.

    https://doi.org/10.1038/nature25787

    • PubMed
    • Google Scholar
12. 1. Douglas ME
    2. Diffley JF

    (2016) Recruitment of Mcm10 to sites of replication initiation requires direct binding to the minichromosome maintenance (MCM) Complex

    Journal of Biological Chemistry 291:5879–5888.

    https://doi.org/10.1074/jbc.M115.707802

    • PubMed
    • Google Scholar
13. 1. Evrin C
    2. Clarke P
    3. Zech J
    4. Lurz R
    5. Sun J
    6. Uhle S
    7. Li H
    8. Stillman B
    9. Speck C

    (2009) A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

    PNAS 106:20240–20245.

    https://doi.org/10.1073/pnas.0911500106

    • PubMed
    • Google Scholar
14. 1. Froelich CA
    2. Kang S
    3. Epling LB
    4. Bell SP
    5. Enemark EJ

    (2014) A conserved MCM single-stranded DNA binding element is essential for replication initiation

    eLife 3:e01993.

    https://doi.org/10.7554/eLife.01993

    • PubMed
    • Google Scholar
15. 1. Gao Y
    2. Cui Y
    3. Fox T
    4. Lin S
    5. Wang H
    6. de Val N
    7. Zhou ZH
    8. Yang W

    (2019) Structures and operating principles of the replisome

    Science 363:eaav7003.

    https://doi.org/10.1126/science.aav7003

    • PubMed
    • Google Scholar
16. 1. Georgescu RE
    2. Langston L
    3. Yao NY
    4. Yurieva O
    5. Zhang D
    6. Finkelstein J
    7. Agarwal T
    8. O'Donnell ME

    (2014) Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

    Nature Structural & Molecular Biology 21:664–670.

    https://doi.org/10.1038/nsmb.2851

    • PubMed
    • Google Scholar
17. 1. Georgescu R
    2. Yuan Z
    3. Bai L
    4. de Luna Almeida Santos R
    5. Sun J
    6. Zhang D
    7. Yurieva O
    8. Li H
    9. O'Donnell ME

    (2017) Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

    PNAS 114:E697–E706.

    https://doi.org/10.1073/pnas.1620500114

    • PubMed
    • Google Scholar
18. 1. Heller RC
    2. Kang S
    3. Lam WM
    4. Chen S
    5. Chan CS
    6. Bell SP

    (2011) Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases

    Cell 146:80–91.

    https://doi.org/10.1016/j.cell.2011.06.012

    • PubMed
    • Google Scholar
19. 1. Ilves I
    2. Petojevic T
    3. Pesavento JJ
    4. Botchan MR

    (2010) Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins

    Molecular Cell 37:247–258.

    https://doi.org/10.1016/j.molcel.2009.12.030

    • PubMed
    • Google Scholar
20. 1. Kang YH
    2. Galal WC
    3. Farina A
    4. Tappin I
    5. Hurwitz J

    (2012) Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis

    PNAS 109:6042–6047.

    https://doi.org/10.1073/pnas.1203734109

    • PubMed
    • Google Scholar
21. 1. Kanke M
    2. Kodama Y
    3. Takahashi TS
    4. Nakagawa T
    5. Masukata H

    (2012) Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components

    The EMBO Journal 31:2182–2194.

    https://doi.org/10.1038/emboj.2012.68

    • PubMed
    • Google Scholar
22. 1. Kaplan DL
    2. O'Donnell M

    (2004) Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes

    Molecular Cell 15:453–465.

    https://doi.org/10.1016/j.molcel.2004.06.039

    • PubMed
    • Google Scholar
23. 1. King GA
    2. Peterman EJ
    3. Wuite GJ

    (2016) Unravelling the structural plasticity of stretched DNA under torsional constraint

    Nature Communications 7:11810.

    https://doi.org/10.1038/ncomms11810

    • PubMed
    • Google Scholar
24. 1. Langston LD
    2. Zhang D
    3. Yurieva O
    4. Georgescu RE
    5. Finkelstein J
    6. Yao NY
    7. Indiani C
    8. O'Donnell ME

    (2014) CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

    PNAS 111:15390–15395.

    https://doi.org/10.1073/pnas.1418334111

    • PubMed
    • Google Scholar
25. 1. Langston LD
    2. Mayle R
    3. Schauer GD
    4. Yurieva O
    5. Zhang D
    6. Yao NY
    7. Georgescu RE
    8. O'Donnell ME

    (2017) Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

    eLife 6:e29118.

    https://doi.org/10.7554/eLife.29118

    • PubMed
    • Google Scholar
26. 1. Langston L
    2. O'Donnell M

    (2017) Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

    eLife 6:e23449.

    https://doi.org/10.7554/eLife.23449

    • PubMed
    • Google Scholar
27. 1. Lee JY
    2. Yang W

    (2006) UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke

    Cell 127:1349–1360.

    https://doi.org/10.1016/j.cell.2006.10.049

    • PubMed
    • Google Scholar
28. 1. Li N
    2. Zhai Y
    3. Zhang Y
    4. Li W
    5. Yang M
    6. Lei J
    7. Tye BK
    8. Gao N

    (2015) Structure of the eukaryotic MCM complex at 3.8 Å

    Nature 524:186–191.

    https://doi.org/10.1038/nature14685

    • PubMed
    • Google Scholar
29. 1. Lõoke M
    2. Maloney MF
    3. Bell SP

    (2017) Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

    Genes & Development 31:291–305.

    https://doi.org/10.1101/gad.291336.116

    • PubMed
    • Google Scholar
30. 1. Lyubimov AY
    2. Strycharska M
    3. Berger JM

    (2011) The nuts and bolts of ring-translocase structure and mechanism

    Current Opinion in Structural Biology 21:240–248.

    https://doi.org/10.1016/j.sbi.2011.01.002

    • PubMed
    • Google Scholar
31. 1. Mayle R
    2. Langston L
    3. Molloy KR
    4. Zhang D
    5. Chait BT
    6. O'Donnell ME

    (2019) Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression

    PNAS 116:798–803.

    https://doi.org/10.1073/pnas.1819107116

    • PubMed
    • Google Scholar
32. 1. Moyer SE
    2. Lewis PW
    3. Botchan MR

    (2006) Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

    PNAS 103:10236–10241.

    https://doi.org/10.1073/pnas.0602400103

    • PubMed
    • Google Scholar
33. 1. Noguchi Y
    2. Yuan Z
    3. Bai L
    4. Schneider S
    5. Zhao G
    6. Stillman B
    7. Speck C
    8. Li H

    (2017) Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model

    PNAS 114:E9529–E9538.

    https://doi.org/10.1073/pnas.1712537114

    • PubMed
    • Google Scholar
34. 1. O'Donnell M
    2. Langston L
    3. Stillman B

    (2013) Principles and concepts of DNA replication in Bacteria, archaea, and eukarya

    Cold Spring Harbor Perspectives in Biology 5:a010108.

    https://doi.org/10.1101/cshperspect.a010108

    • PubMed
    • Google Scholar
35. 1. O'Donnell ME
    2. Li H

    (2018) The ring-shaped hexameric helicases that function at DNA replication forks

    Nature Structural & Molecular Biology 25:122–130.

    https://doi.org/10.1038/s41594-018-0024-x

    • PubMed
    • Google Scholar
36. 1. Parker MW
    2. Botchan MR
    3. Berger JM

    (2017) Mechanisms and regulation of DNA replication initiation in eukaryotes

    Critical Reviews in Biochemistry and Molecular Biology 52:107–144.

    https://doi.org/10.1080/10409238.2016.1274717

    • PubMed
    • Google Scholar
37. 1. Pease PJ
    2. Levy O
    3. Cost GJ
    4. Gore J
    5. Ptacin JL
    6. Sherratt D
    7. Bustamante C
    8. Cozzarelli NR

    (2005) Sequence-directed DNA translocation by purified FtsK

    Science 307:586–590.

    https://doi.org/10.1126/science.1104885

    • PubMed
    • Google Scholar
38. 1. Postow L
    2. Peter BJ
    3. Cozzarelli NR

    (1999) Knot what we thought before: the twisted story of replication

    BioEssays 21:805–808.

    https://doi.org/10.1002/(SICI)1521-1878(199910)21:10<805::AID-BIES1>3.0.CO;2-7

    • PubMed
    • Google Scholar
39. 1. Remus D
    2. Beuron F
    3. Tolun G
    4. Griffith JD
    5. Morris EP
    6. Diffley JF

    (2009) Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing

    Cell 139:719–730.

    https://doi.org/10.1016/j.cell.2009.10.015

    • PubMed
    • Google Scholar
40. 1. Simonetta KR
    2. Kazmirski SL
    3. Goedken ER
    4. Cantor AJ
    5. Kelch BA
    6. McNally R
    7. Seyedin SN
    8. Makino DL
    9. O'Donnell M
    10. Kuriyan J

    (2009) The mechanism of ATP-dependent primer-template recognition by a clamp loader complex

    Cell 137:659–671.

    https://doi.org/10.1016/j.cell.2009.03.044

    • PubMed
    • Google Scholar
41. 1. Smith DE
    2. Tans SJ
    3. Smith SB
    4. Grimes S
    5. Anderson DL
    6. Bustamante C

    (2001) The bacteriophage phi29 portal motor can package DNA against a large internal force

    Nature 413:748–752.

    https://doi.org/10.1038/35099581

    • PubMed
    • Google Scholar
42. 1. Sun B
    2. Johnson DS
    3. Patel G
    4. Smith BY
    5. Pandey M
    6. Patel SS
    7. Wang MD

    (2011) ATP-induced helicase slippage reveals highly coordinated subunits

    Nature 478:132–135.

    https://doi.org/10.1038/nature10409

    • PubMed
    • Google Scholar
43. 1. van Deursen F
    2. Sengupta S
    3. De Piccoli G
    4. Sanchez-Diaz A
    5. Labib K

    (2012) Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

    The EMBO Journal 31:2195–2206.

    https://doi.org/10.1038/emboj.2012.69

    • PubMed
    • Google Scholar
44. 1. van Mameren J
    2. Gross P
    3. Farge G
    4. Hooijman P
    5. Modesti M
    6. Falkenberg M
    7. Wuite GJ
    8. Peterman EJ

    (2009) Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

    PNAS 106:18231–18236.

    https://doi.org/10.1073/pnas.0904322106

    • PubMed
    • Google Scholar
45. 1. Warren EM
    2. Vaithiyalingam S
    3. Haworth J
    4. Greer B
    5. Bielinsky AK
    6. Chazin WJ
    7. Eichman BF

    (2008) Structural basis for DNA binding by replication initiator Mcm10

    Structure 16:1892–1901.

    https://doi.org/10.1016/j.str.2008.10.005

    • PubMed
    • Google Scholar
46. Preprint

    1. Wasserman MR
    2. Schauer GD
    3. O’Donnell ME
    4. Liu S

    (2018) Replisome preservation by a single-stranded DNA gate in the CMG helicase

    bioRxiv.

    https://doi.org/10.1101/368472

    • Google Scholar
47. 1. Watase G
    2. Takisawa H
    3. Kanemaki MT

    (2012) Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS

    Current Biology 22:343–349.

    https://doi.org/10.1016/j.cub.2012.01.023

    • PubMed
    • Google Scholar
48. 1. Yeeles JT
    2. Deegan TD
    3. Janska A
    4. Early A
    5. Diffley JF

    (2015) Regulated eukaryotic DNA replication origin firing with purified proteins

    Nature 519:431–435.

    https://doi.org/10.1038/nature14285

    • PubMed
    • Google Scholar

Author details

1. Lance D Langston

   1. The Rockefeller University, New York, United States
   2. Howard Hughes Medical Institute, New York, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2736-9284

2. Michael E O'Donnell

   1. The Rockefeller University, New York, United States
   2. Howard Hughes Medical Institute, New York, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   odonnel@rockefeller.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9002-4214

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