When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I
2006; Springer Nature; Volume: 25; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601104
ISSN1460-2075
AutoresLouise K. Stanley, Ralf Seidel, Carsten van der Scheer, Nynke H. Dekker, Mark D. Szczelkun, Cees Dekker,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle27 April 2006free access When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124I Louise K Stanley Louise K Stanley DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Ralf Seidel Ralf Seidel (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The NetherlandsPresent address: Biotechnological Centre, University of Technology Dresden, Dresden, Germany Search for more papers by this author Carsten van der Scheer Carsten van der Scheer (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Nynke H Dekker Nynke H Dekker (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Mark D Szczelkun Corresponding Author Mark D Szczelkun DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Cees Dekker Corresponding Author Cees Dekker (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Louise K Stanley Louise K Stanley DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Ralf Seidel Ralf Seidel (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The NetherlandsPresent address: Biotechnological Centre, University of Technology Dresden, Dresden, Germany Search for more papers by this author Carsten van der Scheer Carsten van der Scheer (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Nynke H Dekker Nynke H Dekker (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Mark D Szczelkun Corresponding Author Mark D Szczelkun DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Cees Dekker Corresponding Author Cees Dekker (for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Search for more papers by this author Author Information Louise K Stanley1,‡, Ralf Seidel2,‡, Carsten van der Scheer2, Nynke H Dekker2, Mark D Szczelkun 1 and Cees Dekker 2 1DNA–Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, UK 2(for magnetictweezers experiment), Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands ‡These authors contributed equally to this work *Corresponding authors: (for stopped flow experiments), DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. Tel.: +44 117 928 7439; Fax: +44 117 928 8274; E-mail: [email protected] Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands. Tel.: +31 15 278 6094; Fax: +31 15 278 1202; E-mail: [email protected] The EMBO Journal (2006)25:2230-2239https://doi.org/10.1038/sj.emboj.7601104 Present address: Biotechnological Centre, University of Technology Dresden, Dresden, Germany PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using a combination of single molecule and bulk solution measurements, we have examined the DNA translocation activity of a helicase, the Type I restriction modification enzyme EcoR124I. We find that EcoR124I can translocate past covalent interstrand crosslinks, inconsistent with an obligatory unwinding mechanism. Instead, translocation of the intact dsDNA occurs principally via contacts to the sugar-phosphate backbone and bases of the 3′–5′ strand; contacts to the 5′–3′ strand are not essential for motion but do play a key role in stabilising the motor on the DNA. A model for dsDNA translocation is presented that could be applicable to a wide range of other enzyme complexes that are also labelled as helicases but which do not have actual unwinding activity. Introduction The ATP-dependent translocation of motor proteins along polynucleotides is a fundamental feature of DNA metabolism. One important class of such motors are the helicases (Lohman and Bjornson, 1996). As defined on the basis of characteristic protein motifs, helicases encompass a large enzyme group with diverse genetic roles. For a great many of these proteins, the catalysis of nucleic acid strand separation is key to their translocation mechanism (Soultanas and Wigley, 2001); they are 'helicases' as originally defined (Kuhn et al, 1979). The strand separation activity of these classical helicases is often measured using synthetic polynucleotide substrates. However, there are also many enzymes classified as helicases which fail to unwind such substrates. Occasionally this may be because a vital nucleotide or protein component has not been included in the assay. But more often, the lack of unwinding activity reflects the fact that ssDNA or ssRNA production is not required for the cellular role of the enzyme. A more all-encompassing definition of a helicase is that the protein motifs actually form an energy-coupling module based around RecA-folds that converts the binding and hydrolysis of NTPs into mechanical events on DNA or RNA (Singleton and Wigley, 2002). While the mechanisms of DNA unwinding by helicases have been studied extensively, much less is known about how mechanochemical coupling occurs in other processes catalysed by helicases; for example, the remodelling of polynucleotide structures (e.g., RecG; Singleton et al, 2001) and nucleoprotein complexes (e.g., RSC; Lia et al, 2006). Is nucleic acid strand separation still required for these processes? What mechanistic properties, if any, are shared with the orthodox unwindases? Here, we have addressed these questions using EcoR124I, an Escherichia coli Type I restriction-modification (RM) enzyme that can be defined on the basis of primary amino-acid sequence as a helicase (McClelland and Szczelkun, 2004) and which has DNA translocation activity (Firman and Szczelkun, 2000; Seidel et al, 2004). EcoR124I is a multifunctional, hetero-oligomeric enzyme complex comprising two main components: a core methyltransferase (MTase), which undertakes sequence-specific DNA recognition and modification; and two HsdR subunits, which are loaded by the MTase onto the adjacent nonspecific DNA and which carry out ATP hydrolysis, DNA translocation and endonuclease activities (Szczelkun et al, 1996; Firman and Szczelkun, 2000; Seidel et al, 2004, 2005; McClelland et al, 2005). ATP hydrolysis is coupled to DNA translocation by the superfamily 2 (SF2) helicase motifs in HsdR (McClelland and Szczelkun, 2004). Each HsdR motor translocates independently on the DNA away from the core MTase (Szczelkun et al, 1996; Firman and Szczelkun, 2000; Seidel et al, 2004, 2005; McClelland et al, 2005). As the HsdRs remain bound to the MTase, which in turn remains bound to the EcoR124I binding site, two DNA loops are extruded (van Noort et al, 2004). The MTase–DNA complex is much longer lived than the MTase–HsdR complex so that termination of a translocation event results in HsdR dissociation from both the DNA and the MTase (Seidel et al, 2005). Another motor event is then initiated by the MTase loading a new HsdR molecule. On viral DNA, motor events will fire repeatedly until cleavage is triggered by the collision of two converging HsdRs. Cleavage of the host genome is prevented by attenuation of productive motor events (Makovets et al, 2004; Seidel et al, 2005). No evidence of DNA unwinding by Type I enzymes has been reported using classical oligoduplex helicase assays. However, it is not clear whether the substrates utilised were appropriate to the analysis of a heteroligomeric system such as EcoR124I. One significant problem is that motor activity must be measured in the presence of the MTase loading complex, HsdR has no motor activity in isolation (Firman and Szczelkun, 2000). Since HsdR loading requires the MTase to be bound at a specific dsDNA binding site, it is difficult to imagine how strand separation could be observed using a simple dsDNA substrate. Short oligoduplex substrates, as used in most helicase assays, may also fail because their limited size does not support initiation of productive motor events. Our alternative approach has been to investigate the motor kinetics of EcoR124I on long (>1 kbp) modified DNA using a combination of single molecule and bulk solution techniques (Figure 1). Our results are consistent with a mechanism in which EcoR124I translocates without unwinding along the 3′–5′ strand of intact duplex DNA. Contacts to the 5′–3′ strand play a supplementary 'clamp' role, stabilising the translocating complex. Since Type I enzymes have provided a model system for other helicases, such as the chromatin remodelling factors (Lia et al, 2006), we believe that the dsDNA translocation scheme we present may be generally applicable to other helicase-based motors which do not demonstrate unwinding activity. Figure 1.Approaches for examining the translocation of EcoR124I on modified DNA. (A) Magnetic tweezers assay. A DNA molecule with a single EcoR124I binding site and containing suitable modification(s) is tethered between a magnetic bead and a glass slide. Magnets are used to elongate the DNA by the application of different forces. EcoR124I translocation causes loop extrusion that, in turn, shortens the apparent end-to-end length of the DNA, which can be measured using video microscopy. Due to the close location of the EcoR124I site to the magnetic bead (275 bp for ICLs, 90 bp for gaps) mainly unidirectional DNA translocation towards the modification(s) is probed. Examples are shown of: ICLs (red circle) and accompanying monoadducts (red lollipops); and, a 4 nucleotide gap in the 5′–3′ strand. (B) Triplex displacement assay. A labelled triplex is introduced downstream of an EcoR124I binding site, with modifications to the DNA made in the intervening region. Translocation and collision with triplex leads to displacement and an increase in fluorescence, which can be measured using a stopped-flow fluorimeter. Inhibition of translocation can be inferred from changes to the triplex displacement profiles. Examples are shown of: a 4 nucleotide gap in the 3′–5′ strand; and two abasic groups in 3′–5′ strand introduced by annealing a modified oligonucleotide (red strand) into a gap. Download figure Download PowerPoint Results Is DNA unwinding a prerequisite of DNA translocation by Type I RM enzymes? DNA unwinding by an orthodox helicase is facilitated by ssDNA translocation (Lohman and Bjornson, 1996; Soultanas and Wigley, 2001); intact dsDNA cannot be accommodated by the motor. Accordingly, if interstrand crosslinks (ICLs) are introduced into duplex DNA, an obligate unwindase cannot progress (e.g., DNA unwinding by RecBCD is completely blocked on crosslinked substrates; Figure 2A; Karu and Linn, 1972). But what is the situation for a dsDNA translocase? For one such enzyme, E. coli RuvB, bypass of an ICL has been observed, suggesting that ssDNA does not play an extensive role in translocation (George et al, 2000). It has also been reported that Type I RM enzymes can still cleave crosslinked DNA (Karu and Linn, 1972; Endlich and Linn, 1985), although experimental data were never presented. It was therefore concluded that ICLs have no effect on translocation. However, it has been shown subsequently that Type I enzymes cleave DNA when translocation is impeded (Janscak et al, 1999; also, see below). Therefore, ICLs may actually induce endonuclease activity through inhibition of translocation. To resolve these issues, we analysed EcoR124I on DNA in which ICLs were randomly introduced by treatment with 4′-hydroxymethyl-4,5′,8′-trimethylpsoralen (HTMP) and UV light (Karu and Linn, 1972; Hearst, 1981), using conditions which resulted in either a low or high density of corresponding HTMP monoadducts (see Materials and methods). Figure 2.The effect of DNA ICLs on EcoR124I and RecBCD activity. (A) pLKS3 was linearised with BglII to produce a linear DNA substrate. ∼4 ICLs per DNA were introduced at two HTMP concentrations: either 1 HTMP/dT (high). RecBCD conditions were chosen so that DNA unwinding would occur without DNA cleavage. EcoR124I reactions conditions were chosen that would allow cleavage of circular DNA. DNA substrates and products were separated by gel electrophoresis. (B) Time trace (right panel) recorded in the magnetic tweezers at 20 nM MTase and 160 nM HsdR using a DNA substrate as shown with ICLs introduced at ∼1 ICL/kbp using a low concentration of HTMP (<0.033 HTMP/dT). Multiple motor events are observed over a 90 s window, showing multiple stall events occurring at the same sites on the DNA. (In comparison, on unmodified DNA there is only one stall event corresponding to collision with the bead.) In the left panel is the occupancy of different DNA lengths (all detected lengths of a time trace binned at 10 nm intervals). Peaks in the counts correspond to the full length DNA at ∼2.3 μm (i.e., when the enzyme is not translocating) and correlated stalls at ICLs/adducts (grey lines) and the bead during translocation. If stalling were independent of sequence then the former peaks would not be observed. Download figure Download PowerPoint We first investigated if ICLs could induce cleavage by analysing linear DNA carrying a single EcoR124I site (Figure 2A). Such substrates are normally not cleaved under the reaction conditions used here (Szczelkun et al, 1996). At an average crosslinking density of ∼1 ICL per 1.4 kbp, no cleavage was observed following extensive incubation with EcoR124I (Figure 2A, lanes 5–7). Therefore, neither ICLs nor the accompanying HTMP monoadducts activate EcoR124I endonuclease activity. We next tested circular substrates with a single EcoR124I site that, in contrast to linear DNA, can be cut under the conditions used here (Szczelkun et al, 1996). As has been suggested previously (Karu and Linn, 1972; Endlich and Linn, 1985), ICLs had no effect on the rate or amplitude of DNA cleavage, although elevated monoadduct levels did block EcoR124I binding (Supplementary Figure 1). We also examined the distribution of cleavage sites on circular DNA as a function of increased crosslinking; no significant differences were observed with between 0 and 8 ICLs per DNA (Supplementary Figure 1). Therefore, during cleavage of crosslinked circular DNA, the same collision/cleavage events must be occurring irrespective of multiple ICLs/adducts. To observe directly the effects of ICLs on translocation, we measured motor activity of single EcoR124I molecules using magnetic tweezers (Figure 1A; Seidel et al, 2004, 2005). This setup allows us to monitor the end-to-end distance of a single DNA molecule in real time, with EcoR124I translocation seen as characteristic saw tooth-shaped DNA shortening events (Seidel et al, 2004). We introduced ∼6 ICLs into an 8 kbp linear DNA substrate (Figure 2B) under conditions that produced a low monoadduct density (see Supplementary Methods). While typical profiles on non-crosslinked DNA exhibit translocation events with stalling only observed at the DNA bead (Seidel et al, 2004, 2005), profiles on crosslinked DNA exhibited multiple short temporary stalls within a single translocation event (Figure 2B). There was a strong correlation in stall location between successive events, suggesting that they are dependent upon substrate- and site-specific modifications. In many instances, translocation continued after stalling, showing that the modifications could be bypassed. Given our crosslinking conditions, both randomly distributed ICLs and/or monoadducts could be responsible for these events. Nonetheless, for the longest motor runs observed (representing translocation of ≫1 kbp), at least one ICL will have been overcome on average. We also examined the ATPase activity of EcoR124I on modified and unmodified linear DNA; no differences were observed (data not shown). There is no indication from the single molecule profiles that bypass of DNA damage occurs by DNA release followed by long-range re-association. The same conclusion can be made from the mapping studies (Supplementary Figure 1), where significant changes in motor protein distribution would have been revealed as changes in cleavage site distribution. We confirmed that long-range 'hopping' by EcoR124I is highly unlikely by testing translocation on a DNA catenane (Supplementary Figure 2). Although these data do not completely exclude short-range hopping, this mechanism of bypass seems unlikely for a Type I enzyme as release of the topologically strained expanding loop(s) would most likely result in a rapid diffusion of the HsdR and DNA away from one another, making rebinding unlikely. This is particularly true in the tweezers where separation of the HsdR and loop would be strongly supported by the applied force (1.6 pN in Figure 2B), making any hopping involving a transient disengagement practically impossible. An alternative mechanism of bypass would be for the motor to 'step over' the adduct (see Discussion for detailed definitions of protein step sizes). Given that HTMP crosslinking of thymine residues across a dTA dinucleotide produces a bulky major groove adduct, a protein would most likely need a step size of 3 bp or more to reliably overcome the damage. Since we present evidence in the discussion that EcoR124I steps along DNA in increments of 2 bp or less, we also disfavour this mechanism of bypass. Is there a strand polarity of DNA translocation by Type I RM enzymes? From the data above, strand separation during translocation is highly unlikely and EcoR124I can be classified as a true dsDNA translocase. But how do the HsdRs contact DNA? For the orthodox helicases, the binding and hydrolysis of NTPs has been suggested to drive the opening and closing of a cleft between RecA-like motor domains, which in turn drives motion of ssDNA in one of two polarities—either 5′–3′ or 3′–5′—across the domains (the 'inchworm mechanism'; Soultanas and Wigley, 2001). Thus, helicases can be defined as having a distinct strand polarity (Lohman and Bjornson, 1996). In a similar manner, a dsDNA motor could contact and directionally convey just one strand, the other strand being passively transported. Alternatively, a dsDNA motor could make contacts with both strands. To investigate these alternatives, we introduced ssDNA gaps downstream of an EcoR124I site and measured the effects on DNA translocation using complementary single molecule and bulk solution approaches (Figure 1). For both sets of DNA substrates, the gaps were introduced by modifications of a published method (see Wang and Hays, 2001; Materials and methods; Supplementary Figure 3). One consequence of this method is that the relative locations of the gaps differ from substrate-to-substrate (Supplementary Tables 1–3). However, we show later by the analysis of polyethylene glycol (PEG) spacers that the relative location of an adduct does not affect the outcome (Figure 6A). Translocation on gapped DNA measured using magnetic tweezers EcoR124I motor events on DNA containing a 20 nt gap in one or other strand are shown in Figure 3A. A clear difference between the substrates emerged. (1) On the 3′–5′ modified DNA, only short-range motor events were observed (upper panel). The maximum distance translocated was 1210±20 bp, corresponding to the 1180 bp distance between the EcoR124I site and the gap, with the majority of events terminating almost exactly at this point (Supplementary Figure 4). In comparison, on unmodified DNA, a significant number of translocation events are observed beyond this distance (Supplementary Figure 4). The simplest explanation is that EcoR124I can translocate as far as the 3′–5′ gap but then cannot continue. (2) On the 5′–3′ modified DNA, a significant proportion of events also terminated upon reaching the gap (lower panel). However, in 22±6% of events bypass was observed, with termination occurring downstream of the gap (Supplementary Figure 4). This suggests that EcoR124I can only bypass a 20 nt gap when it is on the 5′–3′ strand. We note that DNA cleavage at the gaps was not observed for either substrate. Figure 3.Single molecule measurements of DNA translocation on gapped DNA. (A) Two time traces recorded in the magnetic tweezers at 20 nM MTase and 160 nM HsdR using the DNA substrate as shown with a 20 nt gap in one or other strand. EcoR124I translocation towards the bead is lost in the thermal noise so the only events observed directly are those towards the gap. The grey lines represent the approximate locations of the gaps. (B) Classification of different motor events as observed at the gap. Broken line represents location of the gap. (C) Statistics of different motors events on each strand as a function of gap length (see Supplementary Methods for details of statistical analysis). Bypass and stall+bypass events were scored together. Download figure Download PowerPoint Four different events could be classified as occurring at a gap (Figure 3B): bypass without visible stalling (bypass); stalling followed by bypass (stall+bypass); stalling followed by dissociation (stalling); and dissociation without visible stalling (dissociation). The statistics of these events were calculated for gaps in each strand, and for variations in gap size (Figure 3C). Bypass and stall+bypass events were scored together. Bypass events were always more efficient when the gap was in the 5′–3′ strand, even when the modification was only a break in the phosphodiester backbone (top panel). This suggests that the principal motor contacts are made to the 3′–5′ strand. This conclusion is reinforced by the stalling probability, which was much greater with gaps on the 3′–5′ strand (middle panel)—a break in the translocated strand would necessarily stall a motor, whereas a break in the non-translocated strand could be overcome by translocation across the intact strand. In contrast, dissociation events were more likely when the gap was in the 5′–3′ strand (lower panel). This suggests that contacts to this strand are more important in stabilising a translocating complex. Given that even a dephosphorylated nick can have a significant effect on the translocation kinetics, it also seems likely that EcoR124I moves in small steps (see below and Discussion). An alternative view of the data is that translocation along the 5′–3′ strand would also lead to an increase in dissociation at 5′–3′ gaps as the motor would dissociate from the end of the DNA. However, in most cases helicase motors tend to be stabilised at DNA ends (e.g., Dillingham et al, 2000), as there are still sufficient contacts between the DNA and the rear of the motor. Data on modified DNA presented below (Figure 6) supports the notion that the 3′–5′ strand plays a more important role than the 5′–3′ strand. Therefore, the overall conclusion we draw from our data is that EcoR124I translocates dsDNA principally by contacts to the 3′–5′ strand, with additional stability coming from contacts to the 5′–3′ strand. We have also obtained further support for 3′–5′ translocation by measuring the directionality of EcoR124I motion at DNA branch points (Louise K Stanley and Mark D Szczelkun, unpublished observations). Is applied force critical to the probability of gap bypass? A prerequisite of measuring translocation by a Type I enzyme in the tweezers assay is that the DNA substrate must be extended by the application of force parallel to the chain (1.6 pN in Figure 3A; Seidel et al, 2004). This is critical to our interpretation of the above data as applied force could effect the motor reaction itself; for EcoR124I, initiation rates and processivity are force dependent (Seidel et al, 2004, 2005). Additionally, applied force could alter the conformational dynamics of the DNA (Blumberg et al, 2005; Sankararaman and Marko, 2005; Yan et al, 2005), which might influence the efficiency of gap bridging. To investigate if applied tension affected gap bypass, we re-examined EcoR124I translocation on the 20 nt 3′–5′ gap substrate at a reduced force (F=0.4 pN, Figure 4A). In contrast to the complete inhibition at 1.6 pN (Figure 3A), gap bypass was now observed in ∼12% of events (Figure 4B). Therefore, DNA tension seems to inhibit gap bypass. How these observations tally with possible translocation mechanisms is discussed below. Figure 4.Force dependence of gap bypass on the 3′–5′ strand. (A) Time trace recorded in the magnetic tweezers at 0.4 pN using the DNA substrate as shown in Figure 3A with a 20 nt gap in the 3′–5′ strand. Enzyme concentrations were 20 nM MTase and 80 nM HsdR. EcoR124I translocation towards the bead is lost in the thermal noise so the only events observed directly are those towards the gap. The grey line represents the estimated location of the gap (1190±40 bp, Supplementary Figure 4). Note that compared to Figure 3A, at the lower force there is an increase in noise and a decrease in apparent distances due to the reduced DNA stretching. (Inset) Detail from the full time trace (dotted box) showing stall events at the gap region and one bypass event. (B) Statistics of 3′–5′gap bypass over 20 nt at different forces. The 1.6 pN data is taken from Figure 3C. Calculation of statistics was carried out as in Figure 3. At least some of the reduction in Pstall at 0.4 pN comes from the increased probability of bypass. There is also an increase in Pdissociation, which might reflect complexes that terminate during bypass of 20 nt ssDNA loops. Download figure Download PowerPoint Translocation on gapped DNA measured using triplex displacement To provide supporting data for the interpretations of the single molecule experiments, we also measured translocation on gapped DNA using triplex displacement (Figure 1B; Firman and Szczelkun, 2000; McClelland et al, 2005; Seidel et al, 2005). We generated 5.6 kbp linear DNA substrates containing an EcoR124I site, a gap site 0.6 kbp downstream, followed by a triplex binding site a further 0.6 kbp downstream (Figure 5A; Supplementary Figure 3). Displacement of a fluorescent triplex by a translocating enzyme can be measured and is preceded by a lag phase, the size of which reflects initiation and translocation events (McClelland et al, 2005). Figure 5.Stopped flow measurements of DNA translocation on gapped DNA. (A) Triplex displacement profiles from the stopped flow experiments. The final solution contains 1 nM DNA (0.5 nM triplex), 30 nM MTase, 300 nM HsdR, and 25 μM ATP. (B) Changes in the triplex displacement profiles with different gap sizes, with ΔTlag=Tlagmodified−Tlagunmodified, where 〈Tlagunmodified〉=16.28±0.14 s (the average of Tlag from intact and nicked DNA, which were identical within error, data not shown). A gap size of zero represents a dephosphorylated nick. Due to the method used to make the gaps (Materials and methods, Supplementary Figure 3 and Table 2), all spacings shown are dephosphorylated except for the 30 nt spacing. To within experimental error, phosphorylated gaps gave identical results (data not shown). (C) Comparison of DNA translocation and gap cleavage using a substrate with a 150 nt 3′–5′ gap. A triplex displacement profile from a hand-mixed radioactive triplex assay (5 nM DNA (2.5 nM triplex), 30 nM MTase and 300 nM HsdR) is compared to the appearance of product DNA cleaved at a gap from an agarose gel assay using 3H-labelled DNA (5 nM DNA, 30 MTase and 90 nM HsdR). Similar traces were obtained for the 5′–3′ gap and the 300 nt spacings (data not shown). (D) ΔTlag values calculated for long gaps (all phosphorylated). All error bars in the figure represent the standard error of the mean calculated from ⩾2 independent experiments. Download figure Download PowerPoint Figure 6.Stopped flow measurements of DNA translocation over DNA adducts. Triplex displacement profiles were recorded using DNA substrates containing the modifications indicated (see Supplementary Table 3). Experimental conditions were as given in Figure 5. (A) ΔTlag values measured on DNA containing two C18 PEG spacers (replacing 6 nt of DNA) in one or other strand. The relative location of the spacers was varied from 601–611 bp downstream of the EcoR124I binding site. The dashed lines represent 〈ΔTlag〉 for each strand. (B) ΔTlag values measured on DNA containing the modifications indicated. All error bars in the figure represent the standard error of the mean calculated from ⩾2 independent experiments. Download figure Download PowerPoint Complete triplex displacement was observed on all substrates, irrespective of gap size or strand polarity (e.g., 30 and 5 nt gaps in Figure 5A). We did not observe irreversible inhibition at longer 3′–5′ spacings as in Figure 3A. Since Pbypass is critically sensitive to DNA tension (Figure 4), the higher efficiency in the triplex assay most likely refle
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