1. Mechanistic Analysis of Local Ori Melting and Helicase Assembly by the Papillomavirus E1 Protein 
Stephen Schuck, Arne Stenlund 
Molecular Cell 
Volume 43, Issue 5, Pages (September 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
The E1 DT Is the Precursor for a Functional E1 DH with Unwinding Activity
(A) An EMSA time course experiment was performed to follow the transition from E1 DT to E1 DH. Eighty nanograms of E1 was incubated with 20 fmol of probe in 100 μl of binding buffer in the presence of 2 mM ATP, as described in Experimental Procedures. Ten microliter 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 nanograms of E1 was incubated with 20 fmol of probe in 100 μl of binding buffer in the presence of 10 ng/μl of E. coli SSB and 2 mM ATP, as described in Experimental Procedures. Ten microliter 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 (A) 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 nanograms of E1 was incubated with 20 fmol of probe in 100 μl of binding buffer in the presence of 2 mM ATP as described in Experimental Procedures. Ten microliter 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 A/T-rich regions are indicated.
(E) The permanganate reactivity in three of the time points (1, 8, and 60 min) in (D) 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 (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 (D–F) (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.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 E1-Induced Permanganate Reactivity on Mutant Ori Templates
(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 (A) (1, 8, and 60 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 (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 (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.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Helicase Domain Mutants Are Defective for the Transition between E1 DT and E1 DH
(A) Three levels (2, 4, and 8 ng) of WT E1 and eight 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 8 ng) of WT E1 and eight 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, Mutated residues that affect the DT to DH transition are shown as space fill.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Helicase Activity and Ori Unwinding by E1 Interface Mutants
(A) Three levels (1, 2, and 4 ng) of WT E1 and seven 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 six 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.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Interface Mutants Arrest Early in the Transition from DT to DH
(A) WT E1, eight 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 nine 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 (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 11–14) 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.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 The E1 BS Is Melted by an Untwisting Mechanism
(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 unnicked, nicked, and ligated templates. The 84 bp ori probe generated by PCR (lanes 1–5), the 84 bp probe with nicks at positions 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 position 7–8 (lanes 1–4) or the A16 probe with nicks at either position 3–4 (lanes 5–8), position 7–8 (lanes 9–12), or position 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.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 A Model for Ori Untwisting by the E1 DT
(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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions