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. Author manuscript; available in PMC: 2012 Sep 2.
Published in final edited form as: Mol Cell. 2011 Sep 2;43(5):776–787. doi: 10.1016/j.molcel.2011.06.026

Mechanistic analysis of local ori melting and helicase assembly by the papillomavirus E1 protein

Stephen Schuck 1, Arne Stenlund 1,*
PMCID: PMC3209524  NIHMSID: NIHMS321563  PMID: 21884978

Summary

Preparation of DNA templates for replication requires opening of the duplex to expose single stranded (ss) DNA. The locally melted DNA is required for replicative DNA helicases to initiate unwinding. How local melting is generated in eukaryotic replicons is unknown, but initiator proteins from a handful of eukaryotic viruses can perform this function. Here we dissect the local melting process carried out by the papillomavirus E1 protein. We characterize the melting process kinetically and identify mutations in the E1 helicase and in the ori that arrest the local melting process. We show that a subset of these mutants have specific defects for melting of the center of the ori containing the binding sites for E1 and demonstrate that these mutants fail to untwist the ori DNA. This newfound understanding of how E1 generates local melting suggests possible mechanisms for local melting in other replicons.

Introduction

Initiation of DNA replication on double stranded (ds) DNA requires the exposure of the two DNA strands for use as templates during DNA synthesis. Because replicative DNA helicases are incapable of initiating unwinding from completely dsDNA molecules, an activity other than the helicase is thought to be required for the initial opening of the duplex. In contrast to DNA helicases, which are ubiquitous and have been studied extensively (Patel and Picha, 2000; Singleton et al., 2007), activities that can generate the initial opening of dsDNA at an origin of replication are rare and poorly understood. The best studied of these proteins, the initiator protein DnaA from E. coli, is responsible for recognition of oriC and local melting, and also assists in loading of the DnaB replicative helicase (Bramhill and Kornberg, 1988; Marszalek and Kaguni, 1994; Mott and Berger, 2007; Mott et al., 2008). Although the mechanism by which DnaA melts DNA is not known, structural data has inspired a model where dsDNA wraps around a helical filament of DnaA, resulting in local melting (Erzberger et al., 2006).

In eukaryotes, an ori melting activity has not been identified. Most likely, this means that the local melting activity resides in some combination of the many proteins that are involved in preparation of the DNA template for initiation of replication. It has been suggested that structural similarities between D. melanogaster ORC and DnaA indicate that ORC could generate local melting in a similar fashion as proposed for DnaA (Clarey et al., 2006; Erzberger et al., 2006). Another candidate for a local melting activity is the MCM helicase in combination with other factors in the pre-replication complex (pre-RC)(Bell and Dutta, 2002). The MCM helicase is loaded onto ori DNA in vitro in a manner dependent on ORC, Cdc6, and Cdt 1, however the DNA is not melted in the process and the MCM ring-structure encircles dsDNA (Evrin et al., 2009; Remus et al., 2009). Since this complex is not an active helicase an activating step is believed to be required involving cell cycle dependent phosphorylation and association with auxiliary factors (Ilves et al., 2010; Sclafani and Holzen, 2007; Stillman, 2005). The exact consequence of the activating step is not known but may involve local melting of the dsDNA and passage of one DNA strand from the inside of the MCM ring to the outside, to generate an MCM helicase that encircles one DNA strand (Yardimci et al., 2010).

The initiator proteins encoded by some eukaryotic viruses are known to have local melting activity. In the papovaviruses, the initiator protein, E1 in papillomaviruses and T-ag in polyomaviruses recognizes the ori, melts the origin locally, and provides the replicative DNA helicase activity (Bullock, 1997; Stenlund, 2003). The helicase activity in these polypeptides has been studied extensively, including the generation of multiple crystal structures of the domains required for this activity (Enemark and Joshua-Tor, 2006; Gai et al., 2004; Li et al., 2003). These initiator proteins form double hexamer (DH) complexes corresponding to the replicative DNA helicase (Borowiec et al., 1990; Fouts et al., 1999; Mastrangelo et al., 1989; Wessel et al., 1992). More recent studies have shown that E1 initially forms a double trimer (DT) complex with the ori DNA. This complex is the precursor for the DH helicase and the prime candidate to generate the local melting activity required prior to formation of the DH helicase (Schuck and Stenlund, 2005; Liu et al., 2007). The DT assembly is dependent on two DNA binding activities. One, in the DNA binding domain (DBD), recognizes four E1 binding sites (E1 BS) in the center of the ori (Chen and Stenlund, 2002; Enemark et al., 2002). The other, located in the helicase domain, includes a β-hairpin structure, and binds non-specifically to the sequences flanking the E1 BS (Schuck and Stenlund, 2005; Liu et al., 2007). Based on biochemical and structural data the DT most likely forms a tube or sleeve that encircles ∼60 bp of ori DNA within which local melting activity, as monitored by permanganate reactivity, can be detected (Enemark and Joshua-Tor, 2006; Liu et al., 2007; Sanders and Stenlund, 2000; Schuck and Stenlund, 2005).

Here we dissect local melting and helicase assembly as carried out by the E1 initiator protein. We find that the E1 DT is responsible for generating local melting which is a pre-requisite for the formation of a functional DH helicase. Using permanganate reactivity assays we characterize local melting kinetically and show that the melting process takes place in clearly defined stages. We identify mutations in the E1 helicase domain that arrest local melting at these stages and show that a likely defect of a subset of these mutants is a failure to untwist the ori DNA. Based on these data we conclude that local melting of the ori DNA is generated by an untwisting mechanism. Our findings also suggest a model for how the E1 β-hairpins structures contribute to DNA untwisting.

Results

The E1 DT is the precursor for a functional E1 DH with unwinding activity

To understand how the DT transitions to a DH we simultaneously monitored formation of the DH by EMSA and unwinding of an ori-fragment, i.e. the generation of ssDNA. In the EMSA time-course (Figure 1A), the DT is present already at the first 30s time point (Figure 1A, lane 2). The DH appears at 8-12 min (Figure 1A, lane 6) and the level of this complex increases over time to ∼50% at 60 min (lane 10 and Figure 1C). The parallel unwinding reaction time course (Figure 1B) was carried out under the same conditions as the EMSA except that the unwinding reactions contained E. coli SSB. In this reaction an ssDNA/SSB complex appeared concurrent with DH formation in Figure 1A (Figure 1B, lane 7 and Figure 1C). Under these conditions ∼30% of the template is unwound.

Figure 1. The E1 DT is the precursor for a functional E1 DH with unwinding activity.

Figure 1

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A. An EMSA time course experiment was performed to follow the transition from E1 DT to E1 DH. Eighty ng of E1 was incubated with 20 fmol of probe in 100μl of binding buffer in the presence of 2 mM ATP as described in Materials and Methods. Ten μl samples were removed at the indicated time points and loaded onto a running EMSA gel. The mobility of the DT and the DH are indicated. Lane 1 contained probe alone.

B. Time course of ori fragment unwinding. Eighty ng of E1 was incubated with 20 fmol of probe in 100μl of binding buffer in the presence of 10 ng/μl of E. coli SSB and 2 mM ATP as described in Materials and Methods. Ten μl samples were removed at the indicated time points and loaded onto a running EMSA gel. The mobility of the ssDNA+SSB complex is indicated.

C. The two time course experiments in Figure 1A and B were quantitated and the fraction of the template present as a DH and unwound were plotted as a function of time.

D. A time course experiment was performed to examine E1 induced permanganate reactivity at the ori over time. Eighty ng of E1 was incubated with 20 fmol of probe in 100μl of binding buffer in the presence of 2 mM ATP as described in Materials and Methods. Ten μl samples were removed at the indicated time points, treated with KMnO4 and processed for analysis by sequencing gel. The position of the E1 BS and the AT rich regions are indicated.

E. The permanganate reactivity in three of the time points (1, 8, and 60 min) in panel C were quantitated and the level of permanganate reactivity at each individual position is indicated by a bar.

F. A permanganate reactivity time course similar to that in panel C using wt E1 and the wt ori was performed with time points taken at 1, 3, 6, and 12 min.

G. A cartoon summarizing ori melting. The data from the permanganate reactivity shown in panels C, D, and E, data from (Schuck and Stenlund, 2007) as well as permanganate reactivity from the bottom strand (data not shown) were combined to provide a schematic view of template melting at the different time points. Permanganate reactivity is indicated as ssDNA.

Local melting corresponds to transient permanganate reactivity

To relate the DH formation and unwinding activity to changes in template structure we performed the same time course experiment in the presence of SSB but now measured permanganate reactivity of the template. KMnO4 can oxidize T-residues that are not base-paired and this modification is susceptible to cleavage with piperidine. A time-course of permanganate reactivity on the viral ori shows initial melting at positions flanking the E1 binding site (E1 BS) at ∼1 min (Figure 1D, lane 2). This early pattern is identical to that observed in the presence of ADP and corresponds to the permanganate reactivity associated with the formation of the DT (Schuck and Stenlund, 2007). At the 8 min time point (lane 3), permanganate reactivity is apparent over the E1 BS, and the reactivity over the A-T rich region is intensified. The novel reactivity over the E1 BS is still visible at 15 min but has disappeared at 30 min (compare lanes 4 and 5). The 30 min pattern persists unchanged for more than 2 hours (data not shown). A time course with shorter intervals revealed that E1 BS melting is present already at 3 min (Figure 1F).

Because only a fraction of the template is unwound, at least two populations of template are present at the 60 min time point, i.e. templates that have been unwound (ssDNA), and templates that have not been unwound. Because ssDNA is highly susceptible to permanganate oxidation and generates very small DNA fragments, unwound DNA is not detected under these conditions (Supplemental Figure 1). The persistent pattern of permanganate reactivity observed beyond 30 min therefore corresponds to templates that have not been unwound. Our interpretation is that this reactivity represents dead-end complexes that fail to proceed to unwinding since they persist well beyond the point where unwinding has ceased (1 hr). The only permanganate reactivity that shows the behavior expected from unwinding, i.e. first the appearance and then the disappearance of reactivity, is the reactivity over the E1 BS, which first appears at 3 min, approximately 10 min prior to when we first observe ssDNA and disappears between 15 and 30 min. We believe that the permanganate reactivity over the E1 BS disappears because these templates are unwound.

By combining these data with similar experiments using the labeled bottom strand (Schuck and Stenlund, 2007) we can derive a pattern of early (∼1 min) permanganate reactivity between pos 6-13 on the left hand side of the E1 BS and between pos 7-10 on the right hand side (Figure 1D and E). A summary of these results is shown in the cartoon in Figure 1G. E1 binds to the ori utilizing the DBD for binding to the E1 BS, and the E1 helicase domain for binding to the sequences flanking the E1 BS. For clarity, only one of the three pairs of E1 molecules in the DT is shown. In the presence of ATP, permanganate reactivity over the E1 BS appears at 3 min, persists past 8 min and then disappears, representing the fraction of the template that is unwound. The fraction of the templates that fail to melt the E1 BS represents dead end complexes.

Local melting can be arrested by mutations in the template

Replacement of the sequences to the left of the E1 BS with a stretch of 16 A-residues results in a template that can form a DT, but is defective for DH formation, for unwinding, and for DNA replication demonstrating that the transition from DT to DH is interrupted on such a template (Schuck and Stenlund, 2007). Furthermore, permanganate reactivity assays using this template gives rise to a pattern similar to the transient permanganate reactivity observed at 8 min with the wt probe (Schuck and Stenlund, 2007). We performed a time-course experiment using the bottom strand of the A16 template (Figure 2A and B). Similar to wt template, reactivity was observed on both sides of the E1 BS at 1 min (Figure 2A, lane 2) and at 8 min, prominent reactivity was apparent over the E1 BS (lane 3). The E1 BS reactivity of the mutant template did not disappear, but persisted with only a slight change in intensity for 60 min (lanes 4-6), indicating that on this template the melting process is arrested with a locally melted E1 BS. This is consistent with the high level of permanganate reactivity on the A16 probe, since the arrest causes accumulation at this point, in contrast to the wt template where E1 BS melting is transient. The unwinding defect for the A16 template explains the persistence of E1 BS melting since unwinding would cause the E1 BS melting to disappear.

Figure 2. E1 induced permanganate reactivity on mutant ori templates.

Figure 2

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A. Wt E1 was incubated with the A16 mutant ori probe as described in Figure 1. At the indicated time points, a sample was removed from the mix and treated with KMnO4, followed by processing for analysis by sequencing gel. The positions of the E1 BS and the A/T rich region are indicated.

B. Three of the time points in panel A (1, 8, and 30 min) were quantitated and the level of permanganate reactivity at each position is indicated.

C. A symmetrical artificial ori was generated by replacing the sequences that flank the E1 BS with 16 T-A bp. The 5′ labeled probe corresponding to the top strand (lanes 1-5) or the bottom strand (lanes 6-10) was used for permanganate reactivity assays. The assays were performed in the presence of 2 mM ADP (lanes 2 and 7, respectively) or in the presence of 2 mM ATP as a time course (3, 8, and 15 min, lanes 3-5, and 8-10, respectively).

D. The permanganate reactivity assays shown in panel C were quantitated and a bar indicates the relative level of reactivity at each position.

E. Permanganate reactivity of the E1 β-hairpin mutants K506A and H507A on the A16 template. The A16 template, labeled on the bottom strand, was incubated in the absence of E1 (lane 1), in the presence of wt E1 (lane 4) or in the presence of the β-hairpin mutants K506A (lane 2) or H507A (lane 3). After 8 min, the sample was treated with permanganate and processed for analysis by sequencing gel.

F. The permanganate reactivity assays shown in panel E were quantitated and a bar indicates the relative level of reactivity at each position.

G. A summary of the events involved in the transition from the DT to DH.

To allow measurement of permanganate reactivity at every nucleotide position we generated an artificial symmetrical ori template in which the sequences flanking the E1 BS only contain T-A bp (Figure 2C and D). In the presence of ADP, E1 gave rise to symmetrical permanganate reactivity at positions 8-12 on both sides of the E1 BS (Figure 2C, lanes 2 and 7), consistent with the idea that these sites of permanganate reactivity result from the attachment of the helicase domain to the ori. In the presence of ATP, the permanganate reactivity appeared over the E1 BS and persisted for at least 15 min, similar to that of the A16 probe (Figure 2C, lanes 3-5 and 8-10).

To verify the involvement of the E1 helicase domain and specifically the β-hairpin in the melting of the E1 BS we tested two adjacent β-hairpin mutants (K506A and H507A) for E1 BS melting using the A16 probe at the 8′ time point (Figure 2E and F). H507A gave rise to permanganate reactivity on the flanks of the E1 BS indicative of attachment but failed to generate permanganate reactivity over the E1 BS (compare lanes 3 and 4). K506A did not give rise to permanganate reactivity (lane 2). This result demonstrates that the β-hairpin, which contacts the DNA on the flanks of the E1BS, is required to generate the permanganate reactivity over the E1 BS, which is occupied by the E1 DBD.

These results provide us with a tentative time-line for template melting and DH formation (Figure 2G). The DT forms rapidly, and in the process generates permanganate reactivity on both sides of the E1 BS, corresponding to the attachment sites for the helicase domains in the DT (Figure 1E). This step requires binding of nucleotide but not hydrolysis. In a process that requires ATP hydrolysis (Supplemental Figure 2), at 2-3 min further permanganate reactivity is detected as melting of the E1 BS. The E1 BS melting reaches a maximum at ∼ 8-10 min and then subsides (Figure 1D, lane 3-6). Onset of DH formation and unwinding (ssDNA-SSB complexes) can be detected around 8-12 min and both increase up to 60 min. The ori mutant A16 is arrested at maximal E1 BS melting, corresponding to ∼8 min (Figure 2A).

Mutations on the surface of the E1 helicase domain

To identify mutations in E1 that arrest the transition from DT to DH we performed a complete surface mutagenesis screen of the E1 helicase domain and generated ∼65 alanine substitutions based on the crystal structures of the E1 helicase domain from BPV E1 (Enemark and Joshua-Tor, 2006) and HPV 11 E1 (Abbate et al., 2004). We generated the mutations in the context of the full length E1 protein, expressed the mutant proteins in E. coli, and purified them to apparent homogeneity. Ten of the mutants either did not give rise to a full-length product or were expressed at very low levels. The remaining ∼55 substitutions were tested in an in vitro DNA replication assay to identify mutants defective in some aspect of DNA replication (see Supplemental Table I). Seventeen of the substitutions showed modest (< 5-fold reduction compared to wt E1) or no defects for in vitro DNA synthesis, while the remaining 38 substitutions either lacked replication activity altogether or had severe defects (reduced >5-fold) for in vitro DNA replication.

Many mutations on the surface of the E1 helicase domain affect the transition from DT to DH

To identify mutations that affected the transition between the E1 DT and the DH, we screened the 38 substitutions defective for in vitro DNA replication for DT and DH formation by EMSA. We identified 10 mutants (I423A, N436A, N444A, S456, N459A, T490A, N494A, D504A, S537A, and K461R) that could form the DT in the presence of ADP, but that failed to form the DH in the presence of ATP. The results of EMSA are shown for wt E1 and 8 of these mutants (I423A, N436A, S456A, N459A, T490A, N494A, S537A and K461R) in the presence of ADP and ATP in Figure 3A and B, respectively. Interestingly, all the mutants with two exceptions (D504A and K461R) were able to form an E1 dodecamer on a very short (32 bp) ori probe demonstrating that they are not defective for oligomerization per se (Supplemental Figure 3). When projected onto the hexameric structure of the E1 oligomerization and helicase domain 9 of the 10 mutant residues reside in the interface between the individual subunits in the hexameric structure, while one mutant (K461R) is located in the central channel (Figure 3C).

Figure 3. Helicase domain mutants are defective for the transition between E1 DT and E1 DH.

Figure 3

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A. Three levels (2, 4, and 8 ng) of wt E1 and 8 helicase domain mutants (I423A, N436A, S456A, N459A, K461R, T490A, N494A and S537A), were tested for E1 DT formation in the presence of ADP by EMSA. Lane 1 contained probe alone.

B. Three levels (2, 4, and 8ng) of wt E1 and 8 helicase domain mutants (I423A, N436A, S456A, N459A, T490A, N494A, S537A, and K461R) were tested for DH formation in the presence of ATP using the 84 bp ori probe. In lanes 1 and 2 wt E1 was incubated in the presence of ADP.

C. A cartoon representation of a monomer (left) and a hexamer (right) of the E1 oligomerization/helicase domain based on the X-ray crystal structure of Enemark and Joshua-Tor, 2007. Mutated residues that affect the DT to DH transition are shown as space fill.

The interface mutants have wt DNA helicase activity but are defective for ori fragment unwinding

Because 9 of the 10 mutants defective for the DT to DH transition are localized at the interface between the subunits in the hexamer structure, we expected that some of these substitutions would affect the DNA helicase activity of E1 and we therefore tested the mutants in an oligonucleotide displacement assay (Figure 4A). Surprisingly, only one mutant, D504A, lacked helicase activity (data not shown), while N436A, had a slight defect (∼2 fold) for DNA helicase activity (lanes 8-10). The rest of the mutants had wt levels of activity, demonstrating that these mutations generally do not affect hexamer formation on ssDNA or the activity of the hexameric helicase, including the binding and hydrolysis of ATP. The exception, D504A, has a defect for ATPase activity (data not shown).

Figure 4. Helicase activity and ori unwinding by E1 interface mutants.

Figure 4

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A. Three levels (1, 2, and 4 ng) of wt E1 and 7 interface mutants (I423A, N436A, S456A, N459A, T490A, N494A, and S537A) were tested for DNA helicase activity using an oligonucleotide displacement assay. The mobility of the ssDNA is indicated. In lane 1, no E1 was added.

B. Three levels (1, 2, and 4 ng) of wt E1 and 6 interface mutants (N436A, T490A, N494A, S456A, N459A, and S537A) were tested for ori fragment unwinding activity in the presence of 10 ng/μl of E. coli SSB. Immediately before loading, the samples were treated with Sarkosyl, which disrupts E1 DNA complexes but not ssDNA-SSB complexes. The migration of the ssDNA-SSB complex is indicated. In lane 1, no E1 was added.

The ori unwinding assay is a comprehensive assay that requires local melting of the template, formation of the DH on the ori, and helicase activity of the DH, resulting in the generation of ssDNA (Figure 4B). The assay conditions differ from the unwinding assay shown in Figure 1B in that we added Sarkosyl to the samples immediately before loading the samples on the gel. This treatment disrupts E1-DNA complexes but not ssDNA-SSB complexes. All the interface mutants showed severe defects for ori fragment unwinding. Four mutants lacked activity altogether (N436A, T490A, N494A), Figure 4B, lanes 5-13 and D504A, (data not shown). The three mutants S456A, N459A, and S537A, lanes 14-22, and N444A, I423A, and K461R, (data not shown) showed >10-fold reduced unwinding activity. Consequently, all the mutants had very severe defects for unwinding. As the majority of these mutant proteins had wt or close to wt helicase activity, the defects of these mutants are likely in a step preceding the assembly of the helicase. We believe that the defect of the interface mutants is not in the ability to physically form a DH but rather reflects a defect in template melting required for functional DH formation and unwinding.

All of the interface mutants are defective early in the transition from DT to DH

To determine where in the DT to DH transition the 10 mutants were arrested, we used the permanganate reactivity time-course assay (Figure 5). We tested the mutants at two time points, 8 and 60 min. If a mutant arrests prior to E1 BS melting we would not expect to see permanganate reactivity associated with the E1 BS at either time point. As shown in Figure 5A, wt E1 as expected, gave rise to a clear permanganate reactivity over the E1 BS at 8′ (Figure 5A, lane 1) and this permanganate reactivity was absent at 60′ as observed previously (Figure 5A, lane 2). Eight of the 10 mutants (I423A, N436A, N444A N459A, T490A, N494A, D504A, S537A) shown in Figure 5A (lanes 3-16 and 19-20) did not give rise to significant permanganate reactivity over the E1 BS at either time-point. Similar results were obtained with the remaining mutant S456A (data not shown) demonstrating that 9 of the 10 mutants arrest in the template melting process prior to the E1 BS melting observed at 8 min. The single mutant that behaved differently, K461R, gave rise to strong permanganate reactivity over the E1 BS at both 8 and 60 min (Figure 5A, lanes 17-18). This pattern is strikingly similar to that observed with the wt E1 on the A16 template (Figure 2A) where prominent E1 BS melting appears at the same time as for the wt template and persists up to the 60 min time point. Since this mutant arrests with the E1 BS melted the arrest point is much later than for the 9 mutants in the interface. One other mutant, D504A gave rise to a slightly anomalous permanganate pattern (lanes 15-16). This mutant failed to show significant T-A melting at either time point.

Figure 5. The interface mutants arrest early in the transition from DT to DH.

Figure 5

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A. Wt E1, 8 interface mutants (I423A, N436A, N444A, N459A, T490A, N494A, D504A and S537A), and K461R were tested in the permanganate reactivity assay at two time points, 8 and 60 min using the wt ori probe labeled on the top strand. The positions of the A/T rich region and the E1 BS are indicated.

B. The 9 interface mutants (S537A, N494A, T490A, N459A, I423A, S456A, N444A, N436A, and D504) were tested at the 8 min time-point in a permanganate reactivity assay using the A16 probe labeled on the bottom strand. As a standard, in lanes 10-13, wt E1 was used in a time course experiment (1, 2, 4, and 8 min) in the presence of ATP. In lane 14, the permanganate reactivity in the presence of ADP was determined.

C. The permanganate reactivity assays in lanes 11-14 in panel B were quantitated and a bar indicates the relative level of permanganate reactivity at each position in the ori sequence.

D. Plasmid untwisting assays were performed using a pUC 19 plasmid template either containing (ori+, lanes 1-9) or lacking (ori-, lanes 10-14) the ori. Time course experiments were carried out by incubating the relaxed plasmids with either wt E1 (lanes 6-9 and 11-13) or K461R (lanes 2-5), in the presence of ATP and topoisomerase I, at room temperature. In lanes 1 and 10 no E1 was added. Samples were removed at the indicated time-points and prepared for agarose gel electrophoresis in the presence of chloroquine to determine the topoisomer distribution. The change in linking number (−ΔLk) for each sample is shown below each lane.

E. Wt E1 (lanes 6-9) and the N436A interface mutant (lanes 2-5) were compared in a time course experiment to measure their ability to untwist an ori plasmid. In lane 1, no E1 was added. The change in linking number (−ΔLk) for each sample is shown below each lane.

To time the melting defect of the 10 interface mutants more precisely, we performed a short time course experiment with the wt E1 using the A16 template either in the presence of ADP (lane 14) or at 1′, 2′, 4′, and 8′ in the presence of ATP (Figure 5B). The permanganate reactivity pattern on the A16 template shows characteristic changes over this time interval (Figure 5B, lanes 10-13). In the presence of ADP, we observe modest permanganate reactivity at pos. 10-13 (Figure 5B, lane 14). After 1′ in the presence of ATP, we observe strong permanganate reactivity only at pos. 3-8 (Figure 5B, lane 13 and Figure 5C). E1 BS melting first appears at 2 min and increases in intensity concomitant with a decrease in intensity of the melting at pos. 3-8 (lanes 10-13). We next compared the pattern generated by the interface mutants at 8 min to the wt time course (Figure 5B, lanes 1-9). D504A (lane 9) shows the earliest arrest point, generating a permanganate pattern similar to wt E1 in the presence of ADP, consistent with an ATPase defect for this mutant and the very weak permanganate reactivity of the T-A stretch (Figure 5A, lanes 15-16). All the other mutants appear to arrest within the first 1-2 minutes, i.e. prior to significant E1 BS melting, (Figure 5B, compare lanes 1-8 to lanes 12-13).

Clearly, K461R arrests significantly later and does not belong in the same category as the other 9 mutants. This mutant most likely arrests at a point immediately preceding the DH formation because the substituted arginine, which projects into the central channel, is too large to allow DH formation. This is consistent with the failure of this mutant to form a dodecamer complex on the short ori probe (Supplemental Figure 3).

Ori plasmid untwisting

To provide a quantitative measure of the level of melting generated by E1 we turned to a plasmid untwisting assay(Dean and Hurwitz, 1991). In these assays a relaxed plasmid is incubated with E1, topoisomerase I, and ATP. Local melting at the ori results in the induction of compensating positive supercoils in the sequences outside the ori. These positive supercoils are removed by the action of topoisomerase I, reducing the average linking number. This change in average linking number can be measured by agarose gel electrophoresis in the presence of chloroquine, after de-proteinization of the DNA. We performed these assays as time-course experiments just as for the permanganate reactivity assays. We established the specificity of the assay by comparing the effect of wt E1 on templates containing or lacking the viral ori (Figure 5D). In the absence of the ori we observed very slight changes in the linking number (ΔLk -0.7 to -0.8), which remained constant over time (Figure 5D, compare lanes 10-14). In the presence of the ori, the changes in linking number were much greater and changed over time. The linking number for wt E1 changed from -0.7 at 2 min to -2.0 at 10 min and to -3.0 at 30 min (compare lane 1 to lanes 6-9), indicating that ∼30 bp are untwisted at the end of the reaction. Analysis of the K461R mutant showed a similar pattern except that this mutant arrests at 10 min, with a maximal change in linking number of ∼-2.0 (compare lanes 3-5 and lane 7). The interface mutants tested in this reaction, such as N436A (Figure 5E, lanes 1-5), showed a maximal change in linking number of ∼-0.8, the equivalent of the untwisting observed at ∼2 min for the wt protein (compare lanes 2-5 with lane 6 in Figure 5E). These results show that the E1 interface mutants are defective in both the ori untwisting and permanganate reactivity assays. Both assays indicate an arrest point for the interface mutants ∼2 min into the DT to DH transition. K461R arrests at ∼8 min in both assays, indicating that the assays measure similar events in spite of the difference in template structure.

The DT is responsible for template melting

The template melting that we observe during the transition from DT to DH could either be generated by the DT, the DH, or by intermediates between the DT and DH. To distinguish between these possibilities we performed a time-course experiment in which we compared permanganate reactivity of E1 complexes that were allowed to proceed to the DH, to E1 complexes that were prevented from forming the DH or potential intermediates by the addition of competitor DNA immediately after DT formation (Supplemental Figure 4). The permanganate activity was virtually identical in the absence and presence of competitor DNA indicating that intermediates in the transition from DT to DH do not play a role in local melting.

In summary, binding of E1 as a DT generates permanganate reactivity on the flanks of the E1 BS through the interaction of the E1 β-hairpins. ATP hydrolysis induces melting that extends from the flanks to the center of the ori, which contains the E1BS. The 9 interface mutants arrest immediately prior to melting of the E1 BS while the K461R mutant and the A16 ori template arrest later, at a point where the E1 BS is melted and just prior to DH formation and unwinding. The melted E1 BS provides the substrate for the DH formation and the active DH unwinds the template to generate ssDNA.

The mechanism of melting

By hydrolyzing ATP the DT extends the permanganate reactivity into the E1 BS in a process that depends on H507 in the β-hairpin (Figure 2E) although the β-hairpin is is positioned on the flanks of the E1 BS. This indicates that the melting of the E1 BS could be indirect and caused by structural changes on the flanks of the E1 BS that are then transferred through the DNA into the E1BS. Such a mechanism would require intact DNA strands since force has to be transmitted through the DNA. To determine whether nicks in the DNA inhibited ori melting we generated ori templates with nicks at specific points and tested these for DT and DH formation (Supplemental Figure 5), for unwinding (Figure 6B) and for permanganate reactivity (Figure 6C) before or after addition of T4 DNA ligase.

Figure 6. The E1 BS is melted by an untwisting mechanism.

Figure 6

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A. The DNA sequence of the 84 bp ori is shown, indicating the positions of the E1 BS, the attachment sites for the E1 helicase domain, and the position of the symmetrical nicks (3-4, 7-8 and 16-17) on both sides of the E1 BS.

B. Comparison of unwinding of un-nicked, nicked, and ligated templates. The 84 bp ori probe generated by PCR (lanes 1-5), the 84 bp probe with nicks at pos. 7-8 generated from annealed oligonucleotides (lanes 6-10) and the nicked probe after ligation (lanes 11-15) were tested for unwinding by wt E1 in the presence of E. coli SSB. In each set, 1, 2, and 4 ng of E1 was used, respectively). In lanes 1, 6 and 11, no E1 was added and lanes 2, 7, and 12 contained boiled probe.

C. Permanganate reactivity of nicked probes. Reactions containing either the wt 84 bp probe with nicks between pos. 7-8 (lanes 1-4), or the A16 probe with nicks at either pos 3-4 (lanes 5-8), pos 7-8 (lanes 9-12) or pos 16-17 (lanes 13-16) were tested for permanganate reactivity at the 8 min time point. For each set, the corresponding ligated probes (lanes 3-4, 7-8, 11-12 and 15-16, respectively) were used as controls.

The wt 84 bp probe was readily unwound by E1 (Figure 6B, lanes 1-5), as was the nicked probe treated with ligase (Figure 6B, lanes 11-15). However, the nicked probe did not give rise to detectable unwinding (Figure 6B, lanes 8-10). The reason for the two bands in lane 7, boiled probe, is that either 1 or 2 SSB tetramers can bind to a probe of this size. These results demonstrate that the nicks abolish unwinding of the ori fragment.

We next measured the permanganate reactivity of probes containing nicks. We first tested the wt probe with nicks at position 7-8 at 8 min, when the E1 BS melting is most prominent (Figure 6C). On the nicked probe, (lane 2), permanganate reactivity appears in the A-T rich region. After addition of ligase, which seals the nick, the E1 BS melting appears (lane 4), as expected, since the ligated probe is identical to our standard wt probe. This result demonstrates that the two types of permanganate reactivity arise by different mechanisms since the nick affects the melting of the E1 BS but not melting of the T6.

We switched to the A16 template, since on this template the E1 BS melting is more prominent and therefore easier to detect. We generated probes with nicks at either the 3-4 or 7-8 position and we observed similar results as with the wt probe (Figure 6C). A nick at either position resulted in loss of E1 BS melting (lanes 6 and 10) and ligation of the nick resulted in the re-appearance of the E1 BS melting (lanes 8 and 12). Both of these nicks are present between the points of attachment of the E1 helicase domain. We next generated nicks outside the points of attachment for the E1 helicase domain. This nick, between pos 16-17, did no longer affect the E1 BS melting (lane 14) indicating that E1 BS melting is only affected by nicks between the attachment sites for the helicase domains, consistent with an untwisting mechanism for E1 BS melting.

Discussion

Local melting at origins of DNA replication remains an enigmatic process for which mechanistic information is lacking. Here we present an analysis of the melting process as carried out by the papillomavirus E1 initiator protein. The papillomavirus replicon represents an attractive system for analysis of local melting because here, in contrast to virtually all other replicons, we have a fair understanding of the architecture of the complex that is responsible for generating local melting. Although we lack a high-resolution structure for the E1 DT bound to DNA, a combination of structural and biochemical analyses demonstrate that E1 binds to the ori most likely forming a sleeve that encircles the ori DNA(Enemark and Joshua-Tor, 2006; Liu et al., 2007; Sanders and Stenlund, 2000; Schuck and Stenlund, 2005). The ∼42 bp of ori sequence where we can detect permanganate reactivity are completely included in the sequences protected by E1 in high resolution footprinting indicating that local melting takes place inside this sleeve(Sanders and Stenlund, 2000).

The sites of earliest permanganate reactivity, affecting a few bp on each side of the E1 BS, are sites where the E1 helicase domain interacts with dsDNA. This interaction is mediated in part by a β-hairpin structure in the E1 helicase domain and depends on K506 at the tip of the β-hairpin, but is not dependent on the adjacent residue H507. Subsequent to DT formation, in an ATP hydrolysis dependent manner, the melting transiently extends into the E1 BS to which the E1 DT is anchored via the E1 DBDs. This extension of the permanganate reactivity is specifically dependent on H507 in the β-hairpin implicating this residue and the β-hairpin in the transient melting of the E1 BS (Figure 2E).

In principle, the melting detected by permanganate reactivity and in the topological assays could be generated by any number of perturbations of the DNA structure. In practice however, the number of plausible models for local melting become very limited if we take into consideration the arrangement of E1 in the DT complex, the involvement of the β-hairpin in melting, the localization of the melted sequences, and the importance of intact DNA strands for melting. The key indicator of productive melting is the permanganate reactivity over the E1 BS. This structural change could either be generated by forces applied to the E1 BS, or by forces applied to other parts of the ori and transmitted through the DNA. In the DT, the E1 BS are bound by the E1 DBD and because the DT is very stable, major rearrangements of the E1 molecules are unlikely (Schuck and Stenlund, 2005). We can also rule out exchange of E1 molecules in the DT based the on the experiments in Supplemental Figure 4. Therefore, melting of the E1 BS is either generated by the E1 DBD, or by force transmitted through the DNA. Our experiments with the nicked templates clearly point to transmission of force through the DNA. Consistent with such a mechanism permanganate reactivity first builds up adjacent to the E1 BS and then spreads over the E1 BS (Figure 5B). We therefore conclude that untwisting of the DNA is a plausible mechanism for melting of the E1 BS.

How such untwisting is generated is more difficult to determine from our data and our model now becomes more speculative. With the two trimers in the DT attached to DNA rotation of the trimers relative to each other could untwist the DNA. However, how such a rotation might be generated is not obvious given what we know about the structural consequences of ATP hydrolysis in E1. If instead of rotating, however, the two trimers are fixed relative to each other the DNA can be rotated inside the DT to generate untwisting of the DNA, as described below (see Figure 7). The linear motion of a pin, which is inserted into a helical groove on the surface of a cylinder, can rotate the cylinder (Figure 7A). In our model, the grooved cylinder represents dsDNA and the pin corresponds to the β-hairpin.

Figure 7. A model for ori untwisting by the E1 DT.

Figure 7

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A. A cartoon illustrating one way that a linear motion can be translated into rotation. Movement of the pins inserted into the helical grooves of the cylinder results in rotation of the cylinder.

B. A cartoon illustrating the movement of the E1 helicase domain β-hairpin in response to ATP binding and hydrolysis (see text for details).

C. A model for how ori untwisting is generated by the E1 DT. See text for details. For simplicity, only two of the three pairs of E1 molecules present in the DT are shown.

A consequence of ATP binding and hydrolysis by E1 and T-ag is a movement of the β-hairpin (Enemark and Joshua-Tor, 2006; Gai et al., 2004) (Figure 7B). This linear movement, due to the helical structure of DNA, could be translated into rotation of the DNA helix if the β-hairpin is inserted into one of the grooves (see Figure 7C). We have shown here that a mutation at the tip of the hairpin (H507A) results in a specific defect for melting of the E1 BS (Figure 2E). A possible model is therefore that the tip of the β-hairpin interacts in one of the grooves on the flanks of the E1 BS (Figure 7C 1, for clarity, only 4 of the 6 E1 molecules in the DT are shown). The β-hairpin 1 would upon ATP hydrolysis move to the left in the left half of the ori, and hairpin 3 would move to the right in the right half of the ori. This motion would untwist the DNA between the two hairpins (Figure 7C 2). The length of the stroke of the β-hairpin from the T-ag structure is ∼17Å (Gai et al., 2004), while in the E1 structure the length of the stroke is ∼5Å (Enemark and Joshua-Tor, 2006), which would correspond to untwisting of ∼5 bp and ∼1.5 bp, respectively. We envision that as hairpins 1 and 3 move to untwist the DNA, the hairpins from the adjacent E1 molecules (2 and 4) interact with the DNA and function as ratchets that hold the DNA while the first hairpins return to the ATP-bound state, thus maintaining the untwisting generated by the hairpin movement (Figure 7C 3). ATP hydrolysis by the E1 molecules 2 and 4 will then result in a second round of untwisting (Figure 7C 4) with either hairpins 1 and 3 or 5 and 6 functioning as the ratchets. In this manner multiple rounds of ATP hydrolysis and hairpin movement could bring about the substantial untwisting that we observe. The time course experiment in Figure 5B shows that after 1-2 min strong permanganate reactivity has accumulated adjacent to the E1 BS (Figure 5B, lanes 12-13). We believe that the E1 DBDs by binding to the E1 BS resist the untwisting that is generated on the flanks of the E1 BS, resulting in the strong permanganate reactivity that builds up adjacent to the E1 BS. Eventually, as sufficient force is applied, the E1 DBDs are released from their binding sites and permanganate reactivity is transferred to the E1 BS (Figure 5B, lane 11 and Figure 7C 5). This results in the recruitment of additional E1 molecules to the complex and formation of the DH (Figure 7C 6).

The ori mutant A16 and the E1 mutant K461R both arrest immediately prior to DH formation as indicated by the inability to form a stable DH on the A16 template or with the K461R protein. K461 resides in the internal cavity in the E1 hexamer structure and the substitution with a significantly larger Arg would reduce the volume of the cavity significantly, indicating a physical defect for DH formation (Enemark and Joshua-Tor, 2006). The defect of the A16 template is more interesting. The important mutations in this template are 6 T-A to A-T changes at pos 12-17 in the left half of the wt ori. The recognition of these 6T-A bp by E1 in the minor groove results in melting at positions 5-17 (Schuck and Stenlund, 2007). Melting at these positions is insensitive to nicks and therefore generated by a mechanism other than untwisting (Figure 6). We believe that this melting is required to partition the two DNA strands for formation of the E1 hexamers.

The model presented above provides an explanation for the defect of the interface mutants. These mutants clearly generate a low level of untwisting on the flanks of the E1 BS but are unable to generate sufficient force to “pop” the E1 DBD off the E1 BS and melt the E1 BS (Figure 5B). We believe that these mutations may affect the ratcheting mechanism that allows untwisting to accumulate. The ability to function as a ratchet would depend on interactions between adjacent E1 molecules, which the interface mutations may destabilize. Without the ratcheting mechanism only a minimal level of untwisting can be generated, which is what we observe for all the interface mutants.

The melting mechanism that we propose for E1 DT could in principle function also for the MCM helicase, which contains β-hairpin structures similar to those found in E1(Fletcher et al., 2003). Because there are differences between E1 and the MCM complex the exact mechanism would have to differ somewhat. The MCM DH is loaded onto dsDNA through interactions with ORC, CDC6, and Cdt 1 and is therefore not anchored to the DNA the way the E1 DT is anchored to the E1 BS (Evrin et al., 2009; Remus et al., 2009). While anchoring is not a requirement for our proposed untwisting mechanism, the fact that the MCM rings encircle ds DNA presents a problem for helicase action and indicates that after local melting of the dsDNA the ring would have to open to allow passage of one DNA strands to the outside of the ring. Interestingly, recent EM data indicates that the MCM complex indeed can exist in various forms including lock-washer and notched conformations that may allow such a strand transfer to occur (Costa et al., 2011). The same or a similar gap may also be involved in the initial loading of the MCM complex onto dsDNA. The closing of this gap would then permit transition of the MCM complex from a melting activity to a functional helicase.

Experimental Procedures

Expression and Purification of E1

Wt E1 and E1 mutants were expressed in E. coli as N-terminal GST fusions and purified by glutathione agarose affinity chromatography. The GST portion was removed by digestion with thrombin and the material further purified by Mono S ion exchange chromatography (Sedman et al., 1997).

EMSA

Four percent acrylamide gels (39:1 acrylamide:bis) containing 0.5 × TBE, lacking EDTA, were used for all EMSA experiments. E1 was added to the probe (∼2 fmol) in 10 μl binding buffer, (20 mM HEPES [pH 7.5], 70 mM NaCl, 0.7 mg/ml BSA, 0.1% NP40, 5% glycerol, 5 mM DTT, 5 mM MgCl2, and 2 mM ATP or ADP. After incubation at RT for 1 hr, the samples were loaded and run for 2 hr at 9V/cm (Schuck and Stenlund, 2005).

Origin unwinding assays

Unwinding assays were performed by incubating E1 with 2 fmol of ori probe in 10 μl of binding buffer (20 mM HEPES [pH 7.5], 70 mM NaCl, 0.7 mg/ml BSA, 0.1% NP40, 5% glycerol, 5mM DTT, 5 mM MgCl2), in the presence of 10ng/μl E. coli SSB and 2 mM ATP and analyzed by EMSA (Schuck and Stenlund, 2005). In the unwinding experiments in Figure 1, the reactions were carried out at RT. In the unwinding experiment in Figure 4B reactions were carried out at 37°C and 0.1% Sarkosyl was added immediately prior to loading of the samples.

Probes

Probes were generated by PCR using primers end labeled with γ-32P-ATP and T4 polynucleotide kinase. Probes were purified by PAGE, eluted by diffusion, and precipitated. The sequence of the wt 84 bp ori probe is shown in Figure 6A.

Permanganate Reactivity Assays

In all permanganate reactivity assays end labeled probe (∼2 fmol) was incubated with 8 ng of wt or mutant E1 in 10 μl binding buffer, (20 mM HEPES [pH 7.5], 70mM NaCl, 0.7 mg/ml BSA, 0.1% NP40, 5% glycerol, 5 mM DTT, 5 mM MgCl2) and 2 mM ATP or ADP at RT. At the indicated time-points KMnO4 was added to a final concentration of 6 mM and reactions incubated for a further 2 min. Cleavage at modified bases was achieved with 1 M piperidine for 30 min at 90°C (Schuck and Stenlund, 2007).

DNA helicase Assays

Oligonucleotide displacement assay were performed by incubating helicase substrate with E1 in a buffer containing 20 mM HEPES (pH 7.5), 5 mM MgCl2, 2 mM DTT, 2 mM ATP, and 0.7 mg/ml of BSA at 37°C for 15 min. After the incubation, SDS was added to 0.1% and the sample was loaded onto 10 % acrylamide gels (29:1 acrylamide: bis)(Liu et al., 2007).

Untwisting reactions

Untwisting reactions were carried out at room temperature in 20 μl reactions containing 20 mM HEPES [pH 7.5], 70 mM NaCl, 10mM MgCl2, 5 mM ATP, 0.7 mg/ml BSA, 5% glycerol, 0.1 % NP40, 4 U topoisomerase I (Invitrogen), 0.5 μg of relaxed plasmid DNA and 700 ng of wt or mutant E1 protein and analyzed by agarose gel electrophoresis in the presence of chloroquine and stained with ethidium bromide (Dean and Hurwitz, 1991).

Supplementary Material

01

Highlights.

Mutations in the E1 helicase domain arrest the local melting

Ori melting is a multi-step process

Local melting at an origin of replication is generated by an untwisting mechanism

Acknowledgments

This work was supported by a grant RO1 AI 072345 to A. S.

Footnotes

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