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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell. Author manuscript; available in PMC 2012 January 8.
Published in final edited form as:
PMCID: PMC3204357
NIHMSID: NIHMS319630

Eukaryotic Origin-Dependent DNA Replication in vitro Reveals Sequential Action of DDK and S-CDK Kinases

Summary

Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding, in vivo studies demonstrate that DDK drives early-firing origin recruitment of Cdc45 before S-CDK activation. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which leading strand DNA polymerases are recruited prior to origin DNA unwinding and RNA primer synthesis.

Introduction

Since the identification of the first defined eukaryotic origins of replication in S. cerevisiae cells (Stinchcomb et al., 1979), a major goal has been to define the molecular mechanisms by which these sequences direct replication initiation. These short (80-120 bp) origins of replication contain an essential, conserved element called the ARS consensus sequence (ACS) that is bound by the eukaryotic initiator, the origin recognition complex (ORC, Bell and Dutta, 2002). With the exception of some embryonic tissues, the initiation of replication in metazoan organisms also occurs at reproducible positions, however, no consensus sequence is associated with these sites (Cadoret and Prioleau, 2010). Although in vitro assays for the initial helicase loading event at a defined origin exist (Remus and Diffley, 2009), the loaded helicases are inactive and assays for their activation and for origin-dependent replication initiation have not been described.

The events of replication initiation are conserved throughout eukaryotes and occur in two temporally separated steps. Helicase loading occurs during G1, when Cdc6 and Cdt1 are recruited by ORC to origin DNA. These factors coordinately load a head-to-head double-hexamer of the Mcm2-7 replicative helicase around the origin DNA (Evrin et al., 2009; Gambus et al., 2011; Remus et al., 2009). The resulting pre-replicative complex (pre-RC) licenses the associated origin, but Mcm2-7 helicases remains inactive until S-phase.

Initiation of replication is triggered by the activation of the S-phase cyclin-dependent kinase (S-CDK) and Dbf4-dependent Cdc7 kinase (DDK) (Labib, 2010). These kinases stimulate binding of Cdc45 and GINS to Mcm2-7, resulting in the formation of the Cdc45-Mcm2-7-GINS (CMG) complex and helicase activation (Ilves et al., 2010). This event is also referred to as pre-initiation complex formation (Sclafani and Holzen, 2007). In S. cerevisiae cells, S-CDK must phosphorylate two proteins, Sld2 and Sld3, to promote DNA replication (Tanaka et al., 2007; Zegerman and Diffley, 2007). Upon phosphorylation, Sld2 and Sld3 bind the BRCT-repeat protein Dpb11 and the formation of this complex facilitates GINS recruitment (Labib, 2010). S-CDK also stimulates formation of the pre-loading complex (pre-LC, Muramatsu et al., 2010), which is an unstable complex between Sld2, Dpb11, Pol ε and GINS that forms independently of DNA. Mcm4 and Mcm6 are the only essential targets for DDK (Randell et al., 2010; Sheu and Stillman, 2010) although how this phosphorylation facilitates subsequent recruitment of Cdc45 and GINS is unclear. Recent data suggest that DDK phosphorylation of Mcm2-7 removes an inhibitory function of the Mcm4 N-terminus (Sheu and Stillman, 2010) and that this event is regulated by at least two additional kinases (Randell et al., 2010). Although their targets are clear, the order of action of S-CDK and DDK has been controversial (Sclafani and Holzen, 2007).

Origin DNA must be unwound to generate the single-stranded DNA (ssDNA) template needed for polymerase function. The ssDNA binding protein RPA associates with origin DNA prior to replication initiation (Tanaka and Nasmyth, 1998; Walter and Newport, 2000). After origin unwinding, Pol α/primase primes DNA synthesis on both strands, while Pol ε and Pol δ elongate the leading and lagging strands, respectively (Burgers, 2009). Although the Mcm10 protein moves with the replication fork and is required to stabilize the large subunit of DNA polymerase alpha (Pol α, Ricke and Bielinsky, 2004; Zhu et al., 2007), whether Mcm10 is involved in the initial recruitment of Pol α or other DNA polymerases to the replisome is unclear. The order of DNA polymerase origin recruitment and how their assembly depends on DNA unwinding also is uncertain.

Using a combination of purified initiation proteins and S. cerevisiae extracts, we describe assays that recapitulate events at replication origins as the cell cycle proceeds from G1 into S phase. In a S-CDK- and DDK-dependent manner, previously loaded Mcm2-7 helicases recruit multiple proteins required for origin activation. These interactions lead to helicase activation, recruitment of replicative DNA polymerases and DNA replication initiation and elongation. Analysis of these assays reveal a preferred order of DDK and S-CDK function and in vivo studies showed DDK is required during G1 for Cdc45 binding at early-firing origins. In addition, we find that the recruitment of the leading and lagging strand DNA polymerases show different requirements for Mcm10 and DNA unwinding.

Results

Recapitulating the G1 to S phase events of replication in vitro

A major obstacle to the recapitulation of eukaryotic DNA replication initiation in vitro is the incompatibility of the cell cycle conditions required for helicase loading (G1) and activation (S). To overcome this hurdle, we simulated the G1 to S phase transition using a combination of S. cerevisiae extracts, similar to the approach used for nucleus- and origin-independent replication using Xenopus egg extracts (Walter et al., 1998). First, we used G1-arrested extract supplemented with purified Cdc6 to load the replicative helicase onto immobilized ARS1 origin DNA (Bowers et al., 2004; Seki and Diffley, 2000). The loaded Mcm2-7 complexes were isolated from the G1 extract and activated by incubation with an S-phase extract (Fig. 1A).

Figure 1
An Assay for Replisome Assembly in vitro

S phase extracts were prepared from cells modified in two ways to enhance their replication capacity. First, these cells contained a temperature-sensitive allele in the DDK catalytic subunit Cdc7 and were arrested at the non-permissive temperature before extract preparation. Thus, the arrested cells are poised for replication initiation with unreplicated DNA but elevated S-CDK levels. To compensate for a lack of DDK activity, we treated loaded Mcm2-7 with purified DDK prior to addition of S phase extract. Second, these cells overproduced Sld2, Sld3, Dpb11, and Cdc45, which are normally expressed at low levels (Ghaemmaghami et al., 2003). Thus, after origin loading in the G1 extract, the Mcm2-7 helicase is exposed to both essential replication-activating kinases and an extract containing a robust source of the proteins required for origin activation.

After sequential treatment of the loaded Mcm2-7 with DDK and S-phase extract, we observed origin association of the helicase activators Cdc45 and Psf2 (a GINS subunit) as well as Mcm10 (Fig. 1B). These associations were dependent on the addition of S-phase extract (Fig. 1B, lanes 1-2), an intact origin sequence (A-B2-, lane 3), and prior Mcm2-7 loading (-Cdc6, lane 4). If the temperature-arrested S phase extract was replaced with extracts prepared from hydroxyurea (HU) or G1-arrested cells overexpressing Cdc45, Dpb11, Sld2 and Sld3 then Cdc45, GINS and Mcm10 failed to associate (Fig. 1B, lanes 5-6). Interestingly, providing additional DDK to these extracts restored recruitment of all three proteins to the HU extract and Cdc45 recruitment to the G1 extract. In contrast, a nocodazole-arrested extract overexpressing the same four proteins (Fig. 1B, lane 7) showed a similar pattern of protein recruitment with or without added DDK, suggesting that when all factors are present, there is no M-phase barrier to replisome assembly. Together, these properties mirrored the hallmarks of origin activation in vivo.

Distinct requirements for Cdc45 and GINS origin recruitment

We investigated the interdependencies of replication protein recruitment to origin DNA (Fig. 2) by immunodepleting individual factors from the S phase extract and assessing the ability of other replication proteins to associate with the origin. In each case, addition of purified forms of the depleted protein (Fig. S1) restored replication protein recruitment, indicating that the depleted extracts remained active and that other essential proteins had not been co-depleted.

Figure 2
Interdependent Recruitment of Replisome Proteins

Analysis of the depleted extracts uncovered distinct requirements for the recruitment of the helicase activating proteins Cdc45 and GINS (Fig. 2). Only Sld3 depletion resulted in a loss of Cdc45 association, although depletion of GINS showed reduced Cdc45 recruitment. In contrast, Cdc45, Sld3, Sld2 and Dpb11 were each required for stable GINS recruitment. Finally, unlike studies of Xenopus Mcm10 (Wohlschlegel et al., 2002), Cdc45, Dpb11 and GINS associated with the origin DNA in the absence of Mcm10 (Fig. 2). In addition, Mcm10 recruitment was eliminated by depletion of any of the other proteins tested.

A Biochemical Assay for Origin-dependent Replication Initiation

Given that helicase-activating proteins were recruited to the origin-containing DNA template, we probed the reaction for the completion of later steps in the replication initiation process. A ~1 kb linear template poorly supported Pol α recruitment and nucleotide incorporation (Fig. 3A and data not shown). In contrast, a larger, 5.9-kb ARS1 plasmid robustly supported both activities (Fig. 3A, B). Reactions containing the plasmid template included six-fold fewer copies of ARS1 (due to less efficient bead attachment) than reactions with the 1 kb linear template (Fig. 3A, compare ORC levels). Nevertheless, the templates showed similar levels of Mcm2-7 loading yet Pol α and replication levels were much higher for the plasmid template. Thus, plasmid DNA was more efficient for helicase loading, polymerase loading, and replication initiation.

Figure 3
Long DNA Templates Supports Polymerase Loading and Replication Initiation

To exclude the possibility that the observed nucleotide incorporation is the result of non-specific repair events, we tested for properties expected for genuine replication products. Nucleotide incorporation was dependent on prior pre-RC formation and ATP hydrolysis (Fig. 3C, lanes 1-3). Examination of the replication products by native agarose electrophoresis revealed that nucleotide incorporation was associated with a shift in the mobility of the plasmid (Fig. 3B), consistent with formation of theta replication intermediates. Replication products synthesized in the presence of BrdUTP migrated at the position expected for heavy:light DNA in a CsCl gradient, indicating that replication was semi-conservative (Fig. 3G). In addition, no reaction products were detected in the presence of aphidicolin, a potent inhibitor of eukaryotic replicative DNA polymerases (Fig. 3C, lane 4), and nucleotide incorporation accumulated for at least 60 minutes of incubation (Fig. 3D). Most importantly, the replication reaction depended on defined origin sequences. DNA templates with an ARS1 origin lacking an ORC binding site (A-B2-) showed dramatically reduced replication (Fig. 3C, lanes 5-11).

DNA length rather than supercoiling or the circular nature of the plasmid DNA was required for replication. Direct comparison of replication using a 7.6 kb ARS1 plasmid (randomly biotinylated) or a linearized version of the same plasmid (biotinylated at one end) showed that the linear template replicated 2-fold more efficiently than its circular counterpart (Fig. 3C, lanes 8-11), due to longer replication products. This finding suggests that the random attachment of the circular DNA to the magnetic bead inhibited replication by impeding replication forks. We determined total nucleotide incorporation and found that ~ 3% of the total plasmid DNA is replicated in the assay. Incomplete Mcm2-7 loading and replication elongation appear to be the primary reasons for the low levels of incorporation (see discussion).

To determine if the over-expression of Cdc45, Dpb11, Sld2 and Sld3 was important for DNA replication, we tested S-phase extracts from cells with endogenous protein levels. These extracts failed to either initiate replication or recruit GINS or Mcm10 (Fig. 3E, lane 2). By adding purified and active forms of the limiting proteins (see Fig. 2) to the S phase extract, we observed that Cdc45, Sld2 and Dpb11 were each limiting for both events (Fig. 3E, F). Together, these data indicate that this assay accurately recapitulates replication initiation, displaying a dependence on a defined origin, the replicative DNA polymerases and multiple essential replication initiation proteins.

DDK and CDK Are Required For Distinct Steps In Origin Activation

We next asked how the DDK and CDK kinases affected replication factor recruitment and replication initiation. We eliminated DDK activity by omitting purified DDK and S-CDK activity by addition of the S-CDK inhibitor Sic1. Both kinases were essential for replication initiation but their loss had distinct effects on replication protein recruitment (Fig. 4). In the absence of DDK, none of the factors examined associated with origin DNA (Fig. 4A-C). In contrast, S-CDK activity was required for Mcm10, Dpb11 and GINS association but not for Sld3 and Cdc45. The association of Cdc45 and Sld3 in the absence of S-CDK suggested that DDK drives the formation of an initial complex (DDK-dependent complex) that is then acted upon in an S-CDK-catalyzed event to recruit Dpb11, GINS and Mcm10. Consistent with a more robust association of Cdc45 with origins upon entry into S phase (Aparicio et al., 1999), salt extraction experiments showed that Cdc45 association is stabilized by the recruitment of the S-CDK dependent factors (Fig. S2).

Figure 4
DDK and S-CDK Are Required For Distinct Stages of Origin Activation

Our findings predict that Sld3 and Cdc45 origin recruitment depends on DDK, however, Sld3 and Cdc45 associate with early origins in G1 (Aparicio et al., 1999; Kamimura et al., 2001; Kanemaki and Labib, 2006), a time when Dbf4 is a target of APC-dependent degradation (Sclafani and Holzen, 2007). To address the role of DDK during the G1 recruitment of Cdc45 and Sld3, we compared their origin association in CDC7 wild type and temperature sensitive (cdc7-4) cells. Due to reduced Cdc45 and Sld3 origin binding at 37°C in wild-type cells (data not shown), we performed this analysis at 25°C (Cdc45) or 32°C (Sld3). Using either ChIP-Chip (Cdc45) or ChIP-PCR (Sld3), we found that association of Cdc45 and Sld3 with early-firing origins during G1 (ARS305, ARS306 and ARS307) was reduced in cdc7-4 cells (Fig. 4D and E). Weak Cdc45 association with some late-firing origins was not reduced by the cdc7 mutation (Fig. 4E, ARS316), potentially due to residual Cdc7 activity at 25°C. Analysis of sites of Cdc45 binding reduced in the cdc7-4 background identified 49 origins, most of which initiate in the first 20% of S phase (Table S1). Thus, DDK is active in late G1 cells and drives the association of Cdc45 and Sld3 with early origins prior to the S-CDK-dependent recruitment of Sld2, Dpb11 and GINS.

Additional experiments support a model in which DDK acts prior to CDK at the origin. We observed the highest levels of replication protein origin association and replication initiation when the loaded helicases were exposed to DDK first, then exposed to CDK in the S-phase extract (Fig. 4A and C, DDK →CDK). Addition of DDK to the S phase extract exposed the loaded helicases to both kinases simultaneously (DDK with CDK) and resulted in lower protein association and initiation. Finally, if loaded helicases were exposed to CDK and S-phase extract followed by CDK inactivation and addition of DDK (CDK →DDK), we observed no replication initiation (Fig. 4C). Intriguingly, under these conditions association of Cdc45, GINS and Mcm10 were dramatically reduced (Fig. 4A and C) even though equivalent DDK phosphorylation of Mcm4 was observed (Fig. 4C, lanes 6 and 7). This suggests that prior exposure to CDK prevents subsequent DDK phosphorylation of Mcm2-7 from driving origin recruitment of Cdc45 or GINS.

Distinct Requirements for Leading and Lagging DNA Polymerase Recruitment

Three DNA Polymerases act at the eukaryotic replication fork but the assembly of these enzymes at the replisome is poorly understood. Because of their affinity for ssDNA templates, DNA polymerase recruitment could require origin unwinding but this has only been addressed for DNA Pol α (Walter and Newport, 2000). Alternatively, DNA polymerases could directly or indirectly interact with loaded helicase even in the absence of DNA unwinding. Mcm10 has been shown to interact with and stabilize Pol α/primase, however, its role in the initial recruitment of DNA polymerases is unknown.

We first asked if Mcm10 was involved in DNA polymerase recruitment. Depletion of Mcm10 dramatically reduced Pol α loading and DNA replication (Fig. 5A and B). These effects were not due to the destabilization of Pol α in the absence of Mcm10 (Ricke and Bielinsky, 2004), because addition of purified Mcm10 restored both events. Mcm10 depletion had little effect on Pol ε recruitment but reduced Pol δ association by half. Notably, addition of purified MBP-Mcm10 stimulated Pol δ recruitment and DNA synthesis compared with the unperturbed extract (Fig. 5A, lanes 7-9, 5B), suggesting that Mcm10 facilitates Pol δ origin recruitment and that Mcm10 was limiting for this event.

Figure 5
Mcm10 is Required for the Recruitment of Pol α and Pol δ to Origin DNA

To investigate the connection between origin unwinding and replisome assembly we monitored association of the ssDNA binding protein RPA with the template DNA. RPA association with the circular template was dependent on ATP hydrolysis (Fig. 6A, +ATPγS), pre-RC formation (Fig. 6A, -Cdc6), and Cdc45 (Fig. 6B). ATPγS was added after DDK phosphorylation of the Mcm2-7 complex. Consistent with DDK functioning in the ATPγS reaction, Sld3 and Cdc45 are recruited to the origin under these conditions (Fig. S4). Thus, Cdc45 and Sld3 do not require DNA unwinding for their recruitment, consistent with studies showing that inactivation of Mcm2-7 ATP binding motifs does not interfere with Cdc45 recruitment (Ying and Gautier, 2005).

Figure 6
ATP Hydrolysis is Required for RPA Loading and for the Loading of a Subset of Polymerases

Because it was added to the reaction after DDK phosphorylation of loaded Mcm2-7, ATPγS could prevent origin unwinding in two ways: inhibition of CDK activity and/or inhibition of Mcm2-7 ATPase activity. Since we knew the effects of CDK inhibition (Fig. 4), we sought conditions in which ATPγS specifically inhibited Mcm2-7. To this end, we exploited a mutant in Cdk1 (Cdk1-AS) that preferentially binds and hydrolyzes modified ATP (Ubersax et al., 2003). We added purified Clb5-Cdk1-AS and a hydrolyzable form of the modified ATP (6-benzyl-ATP) along with ATPγS to the S phase extract (Fig. 6C). Importantly, in these conditions we observed CDK-dependent phosphorylation of Orc6 (Fig. 6C) but only background levels of RPA association with origin DNA. Thus, in these conditions, ATPγS inhibits an event downstream of CDK function required for DNA unwinding, most likely ATP hydrolysis by Mcm2-7.

To determine which replication proteins and DNA polymerases required DNA unwinding for origin recruitment, we assessed replication protein recruitment in the presence of ATPγS, Clb5-Cdk1-AS and 6-benzyl ATP (Fig. 6C). Consistent with the restoration of CDK activity, the CDK-dependent recruitment of Mcm10 and GINS (Psf2) was not blocked under these conditions (Fig. 6C, lanes 4 and 13). Thus, DNA unwinding is not required for Mcm10 or GINS origin association. Even though Mcm10 is present at the origin and is required for the loading of Pol α and Pol δ (Fig. 5), these polymerases were not recruited in the absence of DNA unwinding. In contrast, in the same conditions Pol ε was present at the origin DNA at similar levels as GINS. Both proteins show reduced recruitment in the presence of ATPγS relative to ATP, most likely due to incomplete restoration of S-CDK activity. Thus, our findings support a model in which DNA unwinding is required for Pol α and δ recruitment but that Mcm10 and Pol ε are recruited independently of this event.

Discussion

Although the temporal separation of helicase loading and activation in eukaryotes is critical for preventing genomic stability, the multiple layers of control that prevent the re-replication of chromosomal DNA have made the examination of replication initiation in vitro difficult. To recapitulate origin-dependent replication in vitro, we used two extracts derived from different cell cycle stages to independently drive the G1 and S phase events of DNA replication initiation. Importantly, the helicase activation and replisome initiation observed here show the hallmarks of these events in vivo: they are dependent on origin DNA, previously loaded Mcm2-7, as well as DDK and S-CDK and helicase activating proteins. Furthermore, the reaction supports loading of all three replicative polymerases onto DNA and substantial, semi-conservative duplication of the DNA template. Analysis of these assays revealed different roles for DDK and S-CDK during helicase activation and distinct requirements for leading and lagging strand polymerase recruitment.

Requirements for origin-dependent replication initiation

The in vitro origin-dependent replication assay provides insights into the fundamental requirements for this event. The difficulty of developing such an assay has led to many proposals to explain a lack of success. Because we see replication using a soluble extract and non-nucleosomal DNA templates (data not shown), we can conclude that an intact nucleus (Pasero and Gasser, 2002), chromatin loops (Cayrou et al., 2010) and a defined chromatin state are not required for origin-dependent replication initiation. Unlike E. coli replication (Bramhill and Kornberg, 1988), these studies indicate that eukaryotic origin DNA unwinding is not driven by negative DNA supercoiling, as long linear templates function well (Fig. 3C). Finally, the absence of nucleosomal DNA argues that negative superhelicity stored in nucleosomes is not required for origin-dependent initiation. Although not essential, it is likely that one or more of these factors enhances replication in vivo. In contrast to these non-essential factors, we found that overexpression of Cdc45, Dpb11 and Sld2 (Fig. 3) and the sequential addition of G1 and S phase extracts (Fig. 1) were critical for replication initiation. Consistent with the intra-S-phase checkpoint inhibiting DDK and Sld3 in the HU-arrested extracts (Lopez-Mosqueda et al., 2010; Zegerman and Diffley, 2010), we found that addition of excess DDK in the context of overexpressed Sld3 restored origin association of Cdc45, GINS and Mcm10 (Fig. 1B). Finally, these assays demonstrate that in vitro loaded Mcm2-7 complexes (Randell et al., 2006; Seki and Diffley, 2000) are competent for replication initiation.

The length of the DNA template also contributed to origin-dependent replication initiation (Fig. 3). Studies in Xenopus egg extracts also reported a lack of initiation on short, linear DNA templates, although the reason for this defect was unclear (Edwards et al., 2002). Interestingly, we found that the 1 kb template showed dramatically reduced association of DNA Pol α. The ORC binding site is only 180 bp from the unattached end of the 1 kb template, suggesting that a larger region of ORC-adjacent DNA is required either to unwind origin DNA (required for DNA Pol α recruitment, Fig. 6) or to assemble a pair of complete replisomes.

Although the proportion of DNA that was replicated in the assay was modest (~3% of total circular plasmid DNA replicated), considering the length of the replication products as well as the extent of Mcm2-7 loading, the efficiency of Mcm2-7 activation was much higher. Because on average only 1/4 to 1/3 of the length of the circular plasmid DNA was replicated, the percentage of plasmids that undergo replication initiation is 3-4 times the amount of total DNA replicated (9-12%). Assuming that two Mcm2-7 hexamers are assembled at each origin (Remus et al., 2009), we find that 12-20% of plasmids have loaded Mcm2-7 complexes. Comparing the percentage of plasmids that underwent replication (9-12%) with those that loaded Mcm2-7 (12-20%) suggests that 45% or more of the loaded Mcm2-7 complexes initiate replication in the assay. Modified assays that do not require bead-coupling of the DNA or that improve the extent of Mcm2-7 loading are likely to enhance the extent of replication. Importantly, despite the incomplete replication observed, the strong dependence of the assay on origin DNA and all of the replication initiation proteins tested makes it a powerful tool to investigate their function.

DDK acts before CDK during the initiation reaction

Our findings support a model in which DDK drives the association of Cdc45 and Sld3 with Mcm2-7 prior to CDK action and GINS recruitment. First, we found that DDK but not S-CDK was required for the initial origin recruitment of Cdc45 and Sld3 (Fig. 4) and that addition of DDK to G1 extracts overexpressing limiting replication proteins also drove Cdc45 association (Fig. 1B). Second, our depletion studies are consistent with Cdc45 and Sld3 interacting prior to GINS and Mcm10 (Fig. 2). Third, in vivo studies showed that DDK was required for the previously described (Aparicio et al., 1999; Kamimura et al., 2001; Kanemaki and Labib, 2006) association of Cdc45 and Sld3 with early-firing origins during G1, a time when S-CDK is inactive. Finally, we found that the order of kinase action influenced both replication factor recruitment and replication initiation, with the most robust replication being observed when Mcm2-7 was treated with DDK prior to CDK (Fig. 4). In contrast to some previous studies (Kubota et al., 2003; Takayama et al., 2003), we did not see a requirement for GINS to observe Cdc45 association, although we did see more stable Cdc45 origin association when GINS was present (Fig. 2 and S2). This difference is consistent with increased Cdc45 origin association in S phase relative to G1 (Aparicio et al., 1999) and the GINS-independence of Cdc45 origin binding versus the requirement of GINS for Cdc45 association with origin-adjacent DNA (Kanemaki and Labib, 2006). These phenomena almost certainly reflect interactions before and after the completion of CMG complex assembly (Ilves et al., 2010). Our findings also are consistent with studies indicating Cdc45 and Sld3 require each other for their origin recruitment (Kamimura et al., 2001; Takayama et al., 2003) and S. pombe studies indicating that Sld3 recruitment is dependent on DDK (Yabuuchi et al., 2006). In addition, the lack of Sld3 in the soluble, S-CDK-dependent complex composed of Sld2, Dpb11, GINS and Pol ε (pre-loading complex, Muramatsu et al., 2010) is consistent with the recruitment of these proteins through interaction with origin-associated Sld3.

This order of events has important implications for the control of helicase activation. Loading Cdc45 and Sld3 before S-CDK action would ensure that the S-CDK-dependent interaction between Sld3, Dpb11 and Sld2 (Tanaka et al., 2007; Zegerman and Diffley, 2007) and the associated recruitment of GINS always occurs at origins and not in solution, preventing formation of soluble CMG complexes. The DDK-dependent loading of Cdc45 during G1 is most robust at the earliest firing origins (Fig. 4 and S3), suggesting that Mcm2-7 complexes loaded at these origins are particularly sensitive to levels of DDK phosphorylation. Interestingly, we observed low levels of Cdc45 at a subset of later-firing origins in G1 cells, suggesting that G1 recruitment of Cdc45 is not exclusive to early-firing origins. Finally our studies provide clear evidence that DDK acts during G1 phase. Thus, the primary mechanism preventing helicase activation prior to S phase is the inhibition of S-CDK activity.

The order of DDK and S-CDK function we observe is consistent with findings in cell-free Xenopus egg extracts (Jares and Blow, 2000; Walter, 2000) where it was observed that DDK acts before CDK to drive replication initiation. These studies also observed an inability to initiate if DDK acted after CDK was inhibited. Under these conditions we observed an inability to recruit Cdc45 and GINS, despite similar levels of Mcm4 phosphorylation by DDK (Fig 4A and C). This suggests that exposure of loaded Mcm2-7 to S-CDK prior to DDK inhibits Cdc45 association but not DDK phosphorylation. Because we see reduced but detectable initiation when loaded Mcm2-7 are exposed to DDK and S-CDK simultaneously (Fig. 4C), we propose that mechanisms exist to coordinate the DDK and S-CDK dependent events when both kinases are present. In contrast to these findings, studies of budding yeast DDK and S-CDK function in vivo suggested that DDK could only function for DNA replication after S-CDK has been activated (Nougarede et al., 2000). Although it has previously been suggested that species-specific differences accounted for this discrepancy (Sclafani and Holzen, 2007), our studies suggest that the difference is more likely to be due to different experimental approaches. In particular, the more complex requirements for kinase activity as hundreds of origins initiate during S phase passage in vivo may not reflect the kinase function at individual origins.

Leading and Lagging Strand DNA Polymerase Recruitment

Our studies reveal distinct requirements for origin recruitment of the leading (Pol ε) and lagging strand (Pol α and δ) DNA polymerases and suggest that Pol ε is recruited prior to Pol α and δ at the origin. Two prior in vivo findings support this order of polymerase assembly. First, the observation that Pol α is not required for Pol ε association at stalled replication forks (Masumoto et al., 2000) is consistent with independent association of these factors with the replisome. Second, the formation of a fragile, DNA-independent complex between Pol ε and Sld2, Dpb11 and GINS independently of DNA (Muramatsu et al., 2010) suggests that Pol ε associates with the origin at the same time as these factors. Since only Pol α/primase can initiate DNA synthesis, the prior recruitment of Pol ε would ensure that leading strand DNA polymerases are present prior to synthesis of any RNA primers. Whether there is a mechanism to ensure that Pol δ is present prior to Polα/primase remains to be determined.

Our analysis of Mcm10 function contrasts with earlier studies in yeast and Xenopus. We found that Mcm10 origin recruitment required Sld3, Cdc45, Sld2, Dpb11, and GINS, however, S. cerevisiae studies found that Mcm10 is recruited to origins in G1 (Homesley et al., 2000; Ricke and Bielinsky, 2004). Similarly, studies in Xenopus extracts found that Mcm10 associates with chromatin before S-CDK and DDK are activated (Wohlschlegel et al., 2002). Although we found that Mcm10 is required for replication and DNA polymerase recruitment, both Cdc45 and GINS were recruited in the absence of Mcm10. In contrast, the Xenopus studies found Mcm10 is required for Cdc45 chromatin association. The importance of Mcm10 for Cdc45 DNA association is unclear in yeast cells (Gregan et al., 2003; Ricke and Bielinsky, 2004; Sawyer et al., 2004). The simplest explanation for these discrepancies is the absence of nucleosomal DNA alters the requirements for Mcm10 and Cdc45 recruitment in our assay. Although we may be assessing a subset of Mcm10 functions, the extensive protein requirements for Mcm10 origin recruitment and the requirement of Mcm10 for Pol α and δ recruitment and replication support the functional relevance of our observations. Indeed, Mcm10 is known to move with the replication fork and interact with and stabilize the large subunit of DNA Pol α (Ricke and Bielinsky, 2004; Zhu et al., 2007), all of which are consistent with our observations.

Based on previous work and our investigation of helicase activation factor and polymerase recruitment in this study, we propose a framework for the assembly of the replisome (Fig. 7). Briefly, during late G1 or early S phase, DDK phosphorylates the Mcm2-7 helicase, promoting the stable recruitment of Sld3 and Cdc45. Next, S-CDK-dependent phosphorylation of Sld2 and Sld3 leads to their Dpb11 binding and recruitment of GINS, and Pol ε (most likely as a complex). These proteins then serve to both recruit Mcm10 and activate the Mcm2-7 helicase, which uses ATP hydrolysis to unwind the origin DNA. Pol α and Pol δ can then be loaded on ssDNA leading to the formation of a complete replisome with accessory proteins such as PCNA, Mrc1, and Ctf4.

Figure 7
A Model for the Events Leading to Replication Initiation

The development of an S. cerevisiae in vitro origin-dependent replication assay provides powerful tools to analyze replication in the future. One important goal will be to attribute more specific molecular function to the different initiation proteins and to elucidate the mechanism and regulation of DNA transactions such as origin melting and initial primer synthesis. The ability to substitute seven different purified proteins into corresponding depleted extracts will allow rapid analysis of mutant protein function in vitro and analysis of the corresponding mutants in vivo. In addition, the ease of epitope tagging in budding yeast cells will facilitate the identification of additional proteins that contribute to replication. The development of related assays that use nucleosomal DNA templates will allow direct assessment of the effects of nucleosomes on replication initiation. Finally, the origin-dependence of the assay provides new approaches to assess the interactions between the origin DNA and replication proteins during the initiation process.

Experimental Procedures

Experimental Procedures are described in detail in the Supplementary Information.

Yeast Strains and Plasmid Construction

The S. cerevisiae strains and plasmids used in this study are listed in Tables S2 and S3, respectively.

Protein Purification

Cdc6 and MBP-Mcm10 were purified from E. coli cells. DDK, Clb5-Cdk1-AS, Sld2, Sld3, Dpb11 and GINS were purified from S. cerevisiae cells.

Preparation of ARS1 DNA-Coupled Beads

The 1 kb linear ARS1 DNA template was generated by PCR as described (Tsakraklides and Bell, 2010). Plasmids DNAs were biotinylated and purified using photoprobe (long arm) biotinylation reagent (Vector Laboratories). Linear pARS1/Nco-Nco templates were prepared by restriction digest followed by biotinylation at one end. Biotinylated DNAs were coupled to streptavidin-coated magnetic beads.

Preparation of Whole-Cell Extracts

Yeast cultures were grown to mid-log phase in YP-Glycerol followed by cell cycle arrest and induction of the GAL1,10 expression by galactose. Cells were arrested in G1 by addition of α-factor, in S-phase by incubation at 38°C or addition of HU or at G2/M using nocodazole. Whole-cell extracts were prepared using a SPEX 6870 Freezer/Mill. Extracts were immunodepleted for 1 h at 4°C by incubating with 1/10 volume of antibody-linked agarose beads.

Replisome Assembly and Replication Assays

Replisome assembly and replication assays were performed in three steps: helicase loading, DDK phosphorylation, and replisome assembly/replication. The first two steps were performed as described (Francis et al., 2009) except that the kinase reaction buffer included spermine. For replisome assembly assays, bead-associated protein-DNA complexes were transferred to reaction mixtures containing an ATP-regenerating system and S-phase extract. After incubation, beads were washed and released from the protein-DNA complexes by exposure to UV light. Unless noted, replisome assembly assays were performed with the 1 kb ARS1 linear DNA template. Replication assays were performed as the replisome assembly assays except that step 3 included 200 μM rNTPs and 40 μM dNTPs (including [α-32P]dCTP). After washing beads, associated proteins were released by boiling in Laemmli buffer. Replication products were analyzed by alkaline or native agarose gel electrophoresis. Reactions supplemented with purified proteins contained 300 nM Sld2-Flag, 100 nM Sld3-Flag, 175 nM Dpb11-Flag, 300 nM Cdc45-3HA/3Flag, 225 nM GINS, 35 nM MBP-Mcm10, 800 nM GST-Sic1, or 240 nM Clb5-Cdk1-AS as indicated. Antibodies used for immunoblotting were: Mcm2-7, UM185; ORC, 1108, (Bowers et al., 2004); anti-HA (12CA5); anti-myc (9E10); Dpb11, sc-12004 (Santa Cruz Biotech); Mcm4-Pi, 213/Mcm4-phospho-S82-D83 (Randell et al., 2010); Rfa1, gift from S. Brill (Rutgers University, Piscataway, NJ).

Supplementary Material

Acknowledgments

We thank A. Amon, I. Cheeseman, D. MacAlpine and T. Takara for comments on the manuscript. Rfa1 antibodies were a gift from S. Brill. We thank R. Sinapius and J. Randell for reagents and K. Galani and H. Blitzblau for help with microarrays. This work was supported by NIH grant GM52339 (S.P.B.). S.P.B., S.C. and S.K. are Howard Hughes Medical Institute employees. R.C.H. was supported by a Damon Runyon Cancer Research Foundation fellowship.

Footnotes

Accession Numbers GEO accession number for Cdc45 ChIP-Chip is GSE29646.

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