DNA looping by the SfiI restriction endonuclease1

Article No. mb981967

J. Mol. Biol. (1998) 281, 433±444

DNA Looping by the Sfi I Restriction Endonuclease Lois M. Wentzell and Stephen E. Halford* Department of Biochemistry School of Medical Sciences University of Bristol University Walk, Bristol BS8 1TD, UK

The S®I endonuclease has to interact with two copies of its recognition sequence before it can cleave DNA. To demonstrate that the reaction of S®I on a DNA with two sites involves the formation of a DNA loop, and to characterise the looping interactions on supercoiled and linear DNA, a series of plasmids was constructed with lengths of DNA between two S®I sites varying from 104 to 211 bp. Both supercoiled and linear forms of each DNA were tested as substrates for S®I. The reactions were monitored from the rates of DNA cleavage and from the generation of partially cleaved products, the latter indicating loop disruption before cleavage of both sites. On both supercoiled and linear DNA, the stabilities of the complexes spanning two S®I sites varied in sinusoidal fashion with the distance between the sites, in the manner characteristic of a process governed by the helical periodicity of DNA. In all cases, the looping interaction was stabilised by DNA supercoiling. The sinusoidal variation from S®I reactions on supercoiled DNA at 50 C yielded a helical repeat of about 11.5 base-pairs per turn. # 1998 Academic Press

*Corresponding author

Keywords: DNA communication; DNA ±protein interaction; DNA supercoiling; helical periodicity; restriction-modi®cation

Introduction The S®I endonuclease differs from the ordinary pattern of type II restriction enzymes (Nobbs et al., 1998a). Instead of being a dimeric protein that cleaves both strands of the DNA at a single recognition site (Roberts & Halford, 1993), S®I exists as a tetramer of identical subunits and it has to interact with two copies of its recognition sequence before it can cleave DNA (Wentzell et al., 1995). On a DNA with two S®I sites, one tetramer binds to both sites, looping out the intervening DNA (Szczelkun & Halford, 1996). The nuclease then acts concurrently at four phosphodiester bonds, one in each strand at each site, usually hydrolysing all four before dissociating from the DNA (Nobbs et al., 1998a). The formation of a complex with the protein bridging the sites is essential for its DNA cleavage activity; it has no activity when bound to just one site (Szczelkun & Halford, 1996). S®I thus provides a test system for DNA looping, a key process in many genetic functions (Schleif, 1992; Rippe et al., 1995). Looping between distant DNA sites occurs at the initiation and terPresent address: Lois M. Wentzell, Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK. E-mail address of the corresponding author: [email protected] 0022±2836/98/330433±12 $30.00/0

mination of replication (Kornberg & Baker, 1992), in the control of gene expression (Bellomy & Record, 1990) and in genome rearrangements by site-speci®c recombination and transposition (Chaconas et al., 1996). However, S®I is a particularly favourable system for studies on the mechanism of looping. Not only is its interaction across two recognition sites obligatory for DNA cleavage but also the relative stability of the loop can be assessed from the number of phosphodiester bonds cleaved per turnover. Under adverse conditions such as high salt or high temperature, the life-time of the looped complex is shorter than that needed for the hydrolysis of four phosphodiester bonds and the enzyme then dissociates from the DNA after cutting just one or two bonds (Nobbs & Halford, 1995; Nobbs et al., 1998a). A protein that can form a loop by spanning sites in cis, on the same molecule of DNA, should also be able to bridge sites in trans, on separate DNA molecules. However, the distance in 3D space between two sites on the same DNA is bounded by the separation of the sites along the DNA contour, while sites on different molecules will usually be separated from each other by much longer distances (Rippe et al., 1995). Consequently, interactions in cis are invariably preferred over those in trans (Schleif, 1992). Evidence for the role of physical proximity in 3D space, as opposed to 1D connectivity along the DNA, has come from study# 1998 Academic Press

434 ing looping interactions on catenates containing two interlinked rings of DNA, with one target site on each ring (Adzuma & Mizuuchi, 1989). Any communication that depends on 1D connectivity cannot connect the sites in the separate rings (Szczelkun et al., 1996). Yet the S®I nuclease cleaved recognition sites on two rings of DNA almost as readily as two sites on the same ring, provided the rings were tethered by catenation: without catenation, the single-site rings were cleaved slowly (Szczelkun & Halford, 1996). If two sites on a DNA molecule are separated by a given number of base-pairs, the mean distance between the sites in 3D space will be smaller in the supercoiled form of the DNA than in its relaxed or linear con®gurations (Vologodskii & Cozzarelli, 1996). Supercoiling should thus facilitate looping (Rippe et al., 1995), yet only a limited amount of experimental data are available from which to assess its contribution to looping interactions. To assess this issue, the looping needs to be examined in vitro on both supercoiled and relaxed DNA but relatively few systems allow for such measurements (KraÈmer et al., 1987, 1988). Many systems produce loops only on supercoiled DNA (Kanaar & Cozzarelli, 1992). Several examples of looping on supercoiled DNA have been analysed in vivo (Lee & Schleif, 1989; Law et al., 1993) but it is dif®cult to compare such studies with those on DNA in vitro, on account of the extensive covering of DNA in vivo with proteins (Hildebrandt & Cozzarelli, 1995). In addition, while some of the techniques used to detect looping in vitro on supercoiled DNA can operate with native plasmids (Parker & Halford, 1991), others require mini-circles of <500 bp (KraÈmer et al., 1988). However, mini-circles may take up unusual con®gurations in terms of both twist and writhe (Bates & Maxwell, 1989). In contrast, DNA cleavage reactions by S®I permit the detection of looping in vitro on either supercoiled or linear DNA molecules of virtually any size. If a protein is to form a loop between the two DNA sites, the intervening DNA will need to be bent, and it may also have to be twisted out of its natural helicity, in order to place the requisite faces of the DNA against the DNA-binding surfaces of the protein (Schleif, 1992; Lewis et al., 1996). These factors will have only marginal effects on loops of >300 bp (Bellomy & Record, 1990), on account of the conformational freedom of long DNA chains (Hagerman, 1988). However, DNA bending will engender a progressive reduction in the stability of the loop as the distance between the sites is shortened, while the helical periodicity of DNA should result in a sinusoidal relationship between loop stability and the length of DNA separating the sites. Indeed, the ®rst indication of the existence of looping was the observation that repression of the ara operon was blocked by the insertion of 5 bp between two binding sites for AraC but not by the insertion of 10 bp (Dunn et al., 1984). A sinusoidal response to variations in the length of DNA

Looping by S®I

between two genetic loci has since become diagnostic for looping (Lee & Schleif, 1989; Haykinson & Johnson, 1993; Law et al., 1993). This strategy is applied here to S®I reactions on both supercoiled and linear DNA.

Results and Discussion Plasmids with closely spaced SfiI sites The plasmid pGB1, a 7.6 kb derivative of pBR322 with two S®I sites separated by 1023 bp, has been used before as a substrate for the S®I restriction enzyme (Wentzell et al., 1995; Nobbs & Halford, 1995; Nobbs et al., 1998a). To analyse S®I reactions across different lengths of DNA, a series of plasmids were constructed from pGB1 with shortened distances between the sites (Figure 1). Two derivatives, pW104 and pW211, were obtained by using the ExoIII/S1 method (Henikoff, 1987) to delete the majority of the intervening DNA (each plasmid in this series is denoted by the number of bp between the scissile bonds in the

Figure 1. Plasmids with closely spaced S®I sites. To construct pW104 and pW211, pGB1 was cleaved with MluI and subjected to deletion mutagenesis by endonuclease III/nuclease S1, prior to re-circularisation of the DNA. Further manipulations yielded the other plasmids as indicated (Wentzell, 1997). The two S®I sites on the plasmids are named 1 and 2, in clockwise order.

Looping by S®I

two S®I sites). These were then processed further to create 12 more derivatives. The complete series included three plasmids with sites 110 bp apart (pW104, pW109 and pW113); ten with separations increasing from 149 to 170 bp, mostly in 2 bp steps (pW149 to pW170); one with a longer distance of 211 bp (pW211); and ®nally pGB1 itself with 1023 bp (Figure 1). If the manipulations used to make these plasmids had introduced an intrinsic bend in the intervening DNA, they would be unsuitable for studies on DNA looping. On both supercoiled and linear DNA, looping interactions can be affected by anisotropic bends in the DNA (Kahn & Crothers, 1992; Kremer et al., 1993). A static bend in a DNA fragment can often be detected by an anomalously low electrophoretic mobility in polyacrylamide compared to fragments without a ®xed bend (Hagerman, 1990). When the plasmids made here were digested by S®I and the smaller of the two products (104 to 211 bp) was analysed by electrophoresis, the mobility of the fragment was in every case as expected for that length of DNA (data not shown). Hence, by this criterion, the plasmids do not appear to carry an anisotropic bend in the short length of DNA between the S®I sites. Two additional plasmids used here carried just one of the S®I sites from pGB1; pGB1/S1 has only the ®rst of the two sites, following a 1.9 kb deletion that removed site 2, while pGB1/S2 has the second site, as a result of a 1.8 kb deletion covering site 1 (Wentzell et al., 1995). The S®I sites on pGB1/S1 and pGB1/S2 are, however, distinct from each other in terms of their ¯anking and spacer sequences (the recognition site for S®I, GGCCnnnn#nGGCC (Qiang & Schildkraut, 1984), contains a 5 bp spacer of unde®ned sequence). The activity of a restriction enzyme can be modulated by the sequences ¯anking the site (Taylor & Halford, 1992) and, in the case of S®I, it is also affected by the spacer sequence (Nobbs et al., 1998b). The manipulations of pGB1 that gave rise to pW104 to pW211 changed neither the spacer sequences at either site nor the DNA immediately ¯anking the sites. The plasmids with one S®I site are cleaved by S®I, albeit at slower rates than the two-site plasmid pGB1, by means of bridging interactions between two molecules of the plasmid (Wentzell et al., 1995). However, elevated salt concentrations diminish bridging interactions between S®I sites on separate DNA molecules more severely than looping interactions across sites on the same DNA (Nobbs & Halford, 1995). Consequently, to ensure that the cleavage of the plasmids with two sites was due primarily to looping in cis rather than bridging in trans, all of the reactions described here were done in the presence of 200 mM NaCl, instead of the 50 mM NaCl used in most studies on this enzyme. In addition, the rates reported here on substrates with two S®I sites are those

435 after subtracting the rate on the one-site DNA, pGB1/S1. Utilisation of substrates with closely spaced Sfi I sites The 14 plasmids, pW104 to pW211, together with pGB1, pGB1/S1 and pGB1/S2, were puri®ed to yield radiolabelled preparations containing 590% supercoiled DNA, at the natural superhelicity of Escherichia coli plasmids (Bates & Maxwell, 1993), with 410% open-circle DNA and virtually no linear DNA. Linear forms were generated by using EcoRV to cut each plasmid into two fragments (Figure 1), to yield one DNA of 4.5 kb with no S®I sites and another of 2.3 kb with two centrally located S®I sites. The size of the latter fragment depends on the length of DNA between the S®I sites: with pGB1/S1 and pGB1/S2, it carries one S®I site. Though only one of the EcoRV fragments possesses S®I site(s), reactions on linear substrates were performed by adding S®I nuclease directly to the mixture of products from the EcoRV digests, in order to maintain the same ratio of S®I sites to non-speci®c DNA as the intact plasmids. Following the addition of the enzyme to the DNA, the progress of the reactions was monitored by withdrawing samples at timed intervals and then analysing the DNA by electrophoresis through agarose, to separate the substrate from the cleaved products, before determining the concentrations of each form of the DNA by scintillation counting (Vipond et al., 1995). The reactions were carried out under steady-state conditions, with the S®I enzyme at a lower concentration than the DNA substrate. The action of S®I on each substrate was assessed ®rst by measuring initial rates for the utilisation of the DNA, from the decrease in the concentration of the substrate during the course of the reaction. (The nature of the products of these reactions, cleaved at either one or both sites in either one or both strands, is described below.) As a representative record, Figure 2 shows the utilisation of the DNA with 158 bp between its S®I sites, in both its supercoiled and linear forms, and also the control reactions on supercoiled and linear DNA molecules with one site. The supercoiled form of pW158 was cleaved much faster than either plasmid with one S®I site (Figure 2a). In contrast, linear DNA from pW158 was cleaved much more slowly than the supercoiled DNA, at a rate that was only slightly faster than that with the linear DNA from pGB1/ S1 (Figure 2b). (S®I fails to cleave linearised pGB1/ S2 under these conditions (Nobbs & Halford, 1995): its site is less receptive to S®I than that on pGB1/S1.) S®I reactions were carried out as in Figure 2 on both the supercoiled and the linear forms of all 14 plasmids in the pW104 to pW211 series, and also on pGB1 (Figure 3). The rates at which the supercoiled substrates were consumed during these reaction were very similar: all were cleaved com-

436

Figure 2. Records for utilisation of supercoiled and linear substrates. Reactions in HS buffer at 50 C contained 0.125 nM S®I nuclease and 5 nM DNA from one of the following, as indicated in both panels: pW158 (*); pGB1/S1 (~); and pGB1/S2 (&). For the reactions in a, the DNA substrates were plasmid preparations containing >95% supercoiled DNA. For reactions in b, the DNA substrates were the same plasmids after digestion by EcoRV. Samples were withdrawn from each reaction at the time-points indicated on the x-axis and the DNA in each sample analysed to determine the residual concentration of the DNA substrate.

paratively rapidly, as noted with pW158, at rates in the range from 1.2 to 1.6 minÿ1 (Figure 3a). The linear substrates were, in every case, cleaved at slower rates than the same DNA in its supercoiled state, but the rates with linear DNA varied markedly with the distance between the sites, from 0.08 to 0.55 minÿ1 (Figure 3b). On linear DNA, the fastest rates were obtained on the molecules with the longest separations, 211 or 1023 bp, and the slowest rate on the molecule with the shortest separation, 104 bp. However, at intermediate lengths, the rates oscillated with the distance: separations of 149, 158 and 170 bp gave slower reaction rates than those of 154 or 166 bp.

Looping by S®I

Figure 3. Rates on supercoiled and linear substrates. Each reaction, in HS buffer at 50 C, contained 0.125 nM S®I nuclease and 5 nM DNA from either a supercoiled plasmid with two S®I sites (a) or a linear DNA with two S®I sites (b). Samples were withdrawn from the reactions at timed intervals and analysed to determine the residual concentration of the DNA substrate. Initial rates were determined from the decline in the concentration of the substrate with time; the rates measured with supercoiled and linear pGB1/S1 (Figure 2) were then subtracted from the values for supercoiled and linear substrates, respectively. The rates are given by the column heights (with error bars to indicate standard deviations from >3 measurements) relative to the scales on the y-axes; the scale for a covers larger values than that for b. Each column refers to one of the plasmids (or its linear derivative, after digestion by EcoRV) in the pW104 to pW211 series or to pGB1; the number of bp separating the S®I sites is shown below the relevant column.

The turnover rate for S®I on pGB1 is limited by the dissociation of the product at the end of the reaction (Nobbs et al., 1998a). Since the rates on supercoiled DNA remained virtually constant as the distance between S®I sites was reduced from 1023 to 104 bp, the same process is likely to be

Looping by S®I

rate-limiting on all of the supercoiled substrates tested here. In this case, the steady-state turnover rate will not re¯ect the readiness with which the S®I enzyme generates a DNA loop by binding to both recognition sites. However, the steady-state rates on the linear substrates were all lower than those on the same DNA in its supercoiled state. Hence, at least one step in the reaction pathway must occur at a lower rate on linear DNA than on supercoiled DNA. Moreover, the step that limits the turnover rate on linear substrates appears to re¯ect the looping ability of S®I, since it resulted in rates that oscillated between high and low vales as the distance between the recognition sites was varied. The reaction pathway for S®I on a DNA with two S®I sites starts with the binding of the tetrameric protein to one site, to give ®rst an unlooped DNA ± protein complex. The DNA-bound protein then interacts with the second S®I site, to form the looped complex, before any phosphodiester bonds are hydrolysed (Nobbs et al., 1998a). The conversation of the unlooped to the looped complex is likely to be rapid, faster than the initial binding of the protein to one site, because the protein already bound to one site on the DNA will be closer to the second site than protein in free solution. In addition, the rate for forming the looped complex is unlikely to be limited by the dynamic ¯uctuations in DNA that are needed to juxtapose sites at separate loci in the chain, since these are also rapid (Oram et al., 1997). Nevertheless, the equilibrium between unlooped and looped complexes may still affect the turnover rate for S®I: an equilibrium position favouring the unlooped over the looped form would diminish the steady-state concentration of the latter and thus reduce the ¯ux through the pathway. If so, the oscillations in the turnover rate for S®I on linear substrates would be due to oscillations in the equilibrium constant between unlooped and looped forms. This model indicates that the looped complexes on linear DNA with S®I sites separated by 149, 158 or 170 bp are less stable than those with sites separated by 154 or 166 bp. In contrast, if this equilibration favoured the looped complex, it would have no impact on the turnover rate, which would be determined instead by one or more of the subsequent steps in the pathway. The latter appears to be the case with supercoiled DNA. Supercoiling thus seems to affect the equilibrium between unlooped and looped complexes, driving it towards the latter. Products from supercoiled DNA with two Sfi I sites The agarose gels used above, to separate the DNA substrates from the cleaved products, also revealed the nature of the cleaved products. A supercoiled DNA with two recognition sites for a restriction enzyme can give rise to the following products, which may be separated from each other and from the substrate by electrophoresis: open-cir-

437

Figure 4. Products from S®I reactions on supercoiled DNA. Reactions in HS buffer at 50 C contained 0.125 nM S®I nuclease and 5 nM DNA from one of the following plasmids, as indicated in each panel: pW104 in a; pW211 in b; pW166, pW168 and pW170 in c. a and b, The concentrations of the supercoiled DNA substrate (*), the open-circle form of the DNA (*) and the linear DNA product (&): the three forms are marked next to each line by SC, OC and LIN, respectively. c, The amount of open-circle DNA produced during the reaction, after correcting the amount of contaminating opencircle DNA present in each plasmid preparation at zero time, from pW166 (*), from pW168 ( & ) and from pW170 (&): both x and y scales in c cover smaller values than those in a and b.

438 cle DNA, from nicking the DNA in one strand at either one or both recognition sites; full-length linear DNA, from cutting both strands at one site; ®nally, the two linear DNA products from cutting both strands at both sites (see Figure 1(a) in the preceding paper; Nobbs et al., 1998a). Under optimal reaction conditions, the DNA liberated at the end of each turnover of S®I on supercoiled pGB1 is mainly the ®nal product cut in both strands at both sites. Reactions at the elevated salt concentrations used here also liberate some of the fulllength linear DNA cut at only one site, though none of the open-circle form of pGB1 is released (Nobbs & Halford, 1995). With pGB1, where the S®I sites are separated by 1023 bp, the full-length linear DNA of 7.6 kb can be resolved from the ®nal products of 6.6 and 1.0 kb but, with plasmids where the sites are separated by 100 bp, the fulllength linear DNA of 6.7 kb cannot be resolved from the ®nal product of 6.6 kb. Consequently, the detection of the products from the supercoiled forms of pW104 to pW211 was limited to open-circle DNA, with single-strand breaks at one or both sites, and linear DNA, with double-strand breaks at one or both sites (Figure 4). In contrast to the lack of open circles from pGB1 (Nobbs & Halford, 1995), S®I produced a substantial amount of open-circle DNA from the supercoiled form of pW104 and a smaller amount of open-circle DNA from pW211 (Figure 4a and b). The concentration of open-circle DNA rose during the course of these reactions to maximal values that exceeded the concentration of the S®I enzyme, so this cannot be DNA still bound to the enzyme as a reaction intermediate. Instead, open-circle DNA must sometimes be liberated from the enzyme before it makes a double-strand break. Though the amount of open-circle DNA formed during these reactions decreased as the separation of the S®I sites was increased from 104 to 211 bp, it was found not to be simply related to the inverse of the distance between the sites. Increasing the distance from 166 to 168 and to 170 bp resulted in { The sinusoidal relationship was assessed by ®tting the data in Figure 5a to the function y ˆ A
sin(ow ‡ P) ‡ C, where A is the amplitude of the response, o its frequency (in the same units as w, i.e. bp), P its phase (the amplitude when w ˆ 0) and C a ®xed offset on the y-axis. The ®tting was done by using the non-linear regression algorithm in GRAFIT (Erithacus Software, Slough, UK) to generate the curve from this function that yielded the minimal sum-ofsquares deviations between the curve and all data points. The same procedure was also used to ®t the data in Figure 8a (see below). The function carries the assumption that the oscillations possess constant amplitudes. This will not be the case over a substantial length of DNA, viz. 100 bp (Bellomy & Record, 1990). However, the variations in distance analysed here, from 149 to 170 bp, correspond to two adjacent oscillations, which as thus likely to have similar amplitudes. In this situation, Fourier methods (viz. See Rhodes & Klug, 1980; Law et al., 1993) are inappropriate.

Looping by S®I

an increase in the amount of open-circle DNA rather than a decrease (Figure 4c). The maximal amount of open-circle DNA formed during the S®I reactions on the supercoiled DNA from each member of the pW104 to pW211 series was measured as above and plotted as a function of the number of bp between the sites (Figure 5). As the separation of the S®I sites was increased from 149 to 170 bp, the amount of opencircle DNA varied cyclically with the length of the intervening DNA, in a manner that could be accommodated by the equation for a sine wave (Figure 5a){. Non-linear regression ®tting of these

Figure 5. Open-circle DNA from supercoiled substrates. Reactions, in HS buffer at 50 C, contained 0.125 nM S®I nuclease and 5 nM DNA (typically, 95% supercoiled) from one plasmid in the series pW104 to pW211. Samples were withdrawn from the reactions at timed intervals and analysed as described for Figure 4 to determine the peak concentration of open-circle DNA reached during the course of each reaction. Maxima for the production of open-circle DNA were corrected for the amount of open-circle DNA present at zero time, as a contaminant in the preparations of supercoiled DNA, and are given on the y-axes in both panels. The data points in a show the maxima for open-circle DNA production (with error bars to indicate standard deviations from 53 measurements) from the plasmids with 149 to 170 bp between S®I sites. The line in a is the sine function{ that gave the optimal ®t to the data: this was obtained with A ˆ ÿ0.36 nM, o ˆ 11.5 bp, P ˆ 5.4 rad and C ˆ 0.52 nM. The heights of the columns in b denote the maxima for open-circle DNA production from the plasmids with 104 to 211 bp between S®I sites; the line in b was generated by extrapolation of the sine wave in a. { See footnote on this page.

Looping by S®I

data to the sine function yielded a periodicity of 11.5(0.5) bp. Hence, despite the uniform rates for the consumption of these substrates by S®I (Figure 3a), the fraction of enzyme turnovers that end with the liberation of DNA carrying singlestrand breaks, rather than double-strand breaks, shows the periodic variation expected for a looping interaction. On substrates with two recognition sites, the S®I nuclease has the potential to cleave four phosphodiester bonds during each turnover, sequentially generating enzyme bound DNA cut in one, two, three and four bonds. However, depending on the relative rates for the hydrolysis of the next bond compared to that for the breakdown of the looped complex, the enzyme may dissociate from the looped DNA to liberate partially cleaved products at any stage in this process (see Figure 7 in the preceding paper; Nobbs et al., 1998a). The yield of products liberated from the enzyme with single-strand breaks should therefore increase as the stability of the looped complex decreases: it is a measure of the instability of the looped complex. On supercoiled substrates, S®I sites separated by 149, 160 or 170 bp gave comparatively unstable loops, since these released relatively large amounts of open-circle DNA (Figure 5a). In contrast, separations of 154 and 166 bp gave looped complexes with enhanced stabilities, which resulted in relatively little opencircle DNA. The periodic variation in loop stability is a re¯ection of the helical repeat of DNA. Its helicity will determine whether or not the two sites are appropriately oriented with respect to each other around the axis of the DNA, so as to allow the enzyme to bind to both sites without perturbing the twist of the intervening DNA (Schleif, 1992). When the sine wave obtained by ®tting the data from pW149 to pW170 was extrapolated to encompass the outlying members of the pW104 to pW211 series, it matched the data from pW211 (Figure 5b). However, the plasmids with short spacings of <113 bp no longer followed the sinusoidal relationship and instead gave maximal amounts of opencircle DNA that increased linearly as the site separation was shortened from 113 to 104 bp. The deviation from the sinusoidal relationship is due to the energetics for forming a small loop of DNA being dominated by DNA bending rather than twisting (Shore & Baldwin, 1983). Products from linear DNA with two SfiI sites The procedure used to measure the formation of products from linear substrates carrying two S®I sites is exempli®ed with DNA from pW168 (Figure 6). EcoRV cleaves pW168 into two fragments; one of 4453 bp that has no S®I sites and one of 2339 bp with two sites. The only products from S®I reactions on the 2339 bp DNA that can be detected on agarose are those with double-strand breaks at either one or both sites: the electrophoretic mobility of a linear duplex is not affected by single-strand nicks. Cleavage of both S®I sites on

439

Figure 6. Reactions at two S®I sites on linear DNA. The plasmid pW168 is cleaved by EcoRV to two fragments: one of 2339 bp that has two S®I sites, named 1 and 2, and one of 4453 bp with no S®I sites. a, The position of the S®I sites on the 2339 bp fragment (hatch marks on the double-stranded DNA) and the products formed by cleaving this DNA at site 1, at site 2 and at both sites 1 and 2; fragment sizes (in bp) are indicated above each DNA. b, An agarose gel carrying samples withdrawn at various times after initiating a reaction of S®I nuclease (0.5 nM) on EcoRV-cleaved pW168 (5 nM) in HS buffer at 50 C. The time of withdrawal (in min) is noted above each lane; except for the 168 bp product, all DNA fragments are noted on the right of the gel (in bp).

this DNA yields three products, of 763, 168 and 1408 bp. However, a reaction at just one site yields one of these ®nal products, either 763 or 1408 bp, and a distinctive partial product: 1576 bp from a reaction at site 1 alone, 931 bp from site 2 alone (Figure 6a). Due to the slow rate at which S®I cleaves linear DNA with closely spaced S®I sites (Figures 2b,3b), elevated S®I concentrations were needed to convert the majority of the linear substrate to the various products noted above. Samples were taken from such reactions at various times and analysed by electrophoresis through agarose, to separate the two partial products from the substrate and all three ®nal products (Figure 6b): the concentration of each was measured, except for the 168 bp DNA, which migrated off the gel. The fraction of the DNA cleaved only at site 1 was measured from the concentration of the 1576 bp partial product, but the 763 bp ®nal product can arise from reactions at

440

Figure 7. Products from S®I reactions on linear DNA. Reactions in HS buffer at 50 C contained 0.5 nM S®I nuclease and 5 nM EcoRV-cleaved DNA from one of the following plasmids, as indicated in each panel: pW166 in a: pW168 in b; pW170 in c. Samples were withdrawn from the reactions at the times indicated on the x-axes and analysed as described for Figure 6 to determine the concentrations of the linear DNA substrate with two S®I sites (*) and the following products, as indicated on the right of each panel: product cleaved only at site 1 (&); product cleaved only at site 2 (*); product cleaved at both sites 1 and 2 ( & ).

either site 1 alone or at both sites 1 and 2: the fraction cut at both sites was assessed from the difference the concentrations of the 763 bp and the 1576 bp products. Similarly, the amounts of the 931 bp partial product and the 1408 bp ®nal pro{ See footnote on p. 438.

Looping by S®I

duct gave the fractions cleaved at site 2 alone and at sites 2 and 1 together. Records of this procedure are shown, not only for the DNA with sites 168 bp apart but also for those with separations of 166 and 170 bp (Figure 7). The reaction of S®I on a linear DNA with two sites 166 bp apart might have been expected to progress in sequential stages, generating ®rst the products cut at one site and only later the products cut at both sites, but this was not observed. Instead, the substrate was partitioned into three sets of products: the majority of the DNA was converted directly to the products cut at both sites 1 and 2, while a smaller fraction was cut only at site 1 and an even smaller fraction just at site 2 (Figure 7a). The initial rate for forming the doubly cut product was faster than that for either singly cut product, thus excluding the possibility that the latter are on the pathway to the DNA cut at both sites. The failure of the singly cut products to proceed to the doubly cut DNA can be accounted for by the rates at which S®I cuts linear DNA with one recognition site: the linear form of pGB1/S1, carrying just site 1, was cleaved very slowly while linear DNA from pGB1/S2, with only site 2, was not cleaved at all (Figure 2b). The difference in the susceptibilities of sites 1 and 2 to S®I also accounts for why more of the singly cut products came from reactions at site 1 rather than site 2. The same test was applied to the EcoRVlinearised DNA from each member of the pW104 to pW211 series. In every case, only a small fraction of the DNA was partitioned into the product cut only at site 2 but the relative amounts of DNA cleaved at 1 alone or at both 1 and 2 differed among these substrates. The DNA with sites 166 bp apart yielded more of the doubly cut product than the product cut only at 1 (Figure 7a). The DNA with sites separated by 168 bp gave similar yields of these two products (Figure 7b). Sites 170 bp apart gave less of the doubly cut product than that from 1 alone (Figure 7c). When analysed across the range of site separations from 149 to 170 bp, the yield of the doubly cut product from the linear substrates showed a sinusoidal dependence on the distance between the sites (Figure 8a): ®tting to the sine function{ yielded a periodicity of 10.9(0.5) bp. The yields of the doubly cut product from the three substrates with short spacings between the S®I sites, 104, 109 and 113 bp, varied among themselves in accordance with this periodicity but, in all three cases, the yields were lower than those expected from extrapolation of the sine wave (Figure 8b). Helical periodicity The sinusoidal function from the linear DNA substrates appears to be the inverse of that with the supercoiled substrates: the maxima in Figure 8a, at separations of 154 and 166 bp, correspond to the minima in Figure 5a. However, the reactions on the supercoiled substrates were assessed from the

441

Looping by S®I

Figure 8. Doubly cut DNA from linear substrates. Reactions, in HS buffer at 50 C, contained 0.5 nM S®I nuclease and 5 nM EcoRV-cleaved DNA from one plasmid in the series pW104 to pW211. Samples were withdrawn from the reactions at timed intervals and analysed as in Figure 6 to determine, at the end-point for each reaction (Figure 7), the concentrations of the DNA products cut at site 1, at site 2 and at both sites 1 and 2. The latter are given on the y-axes in both panels. The data points in a show the amounts of DNA cut at both 1 ‡ 2 (with error bars to indicate standard deviations from 53 measurements) from the plasmids with 149 to 170 bp between S®I sites. The line in a is the sine function{ that gave the optimal ®t to the data: this was obtained with A ˆ 1.1 nM, o ˆ 10.9 bp, P ˆ 1.1 rad and C ˆ 2.2 nM. The heights of the columns in b denote the yields of DNA cut at 1 ‡ 2 from the plasmids with 104 to 211 bp between S®I sites: the line in b was generated by extrapolation of the sine wave in a. { See footnote on page 438.

Several studies have shown that the helical repeat of DNA in solution is close to 10.5 bp/turn (Rhodes & Klug, 1980; Shore & Baldwin, 1983; Tullius & Dombroski, 1985), somewhat less than the values of 11.5(0.5) and 10.9(0.5) bp per turn determined from S®I reactions on supercoiled and linear DNA, respectively (Figures 5a, and 8a). However, the previous studies had been carried out at temperatures in the range from 20 to 37 C, in buffers containing 450 mM salt. In contrast, the S®I reactions were done at 50 C, the optimum for this enzyme (Qiang & Schildkraut, 1984; Nobbs et al., 1998a), in buffers containing 200 mM NaCl. The elevated salt concentration should reduce the helical repeat but this will be a minor effect (Taylor & Hagerman, 1990; Rybenkov et al., 1997), while the raised temperature should increase in the repeat. In addition, the repeat of 10.5 bp/turn refers to linear DNA. The repeat on negatively supercoiled DNA should be higher than this because at least part of its linking de®ciency will emerge as a reduced twist, as opposed to writhe (Bates & Maxwell, 1993). Looping interactions on supercoiled DNA in vitro, by the lac repressor at room temperature and by the Hin recombinase at 37 C, yielded repeats of 11.0 and 11.2 bp/turn, respectively (KraÈmer et al., 1988; Haykinson & Johnson, 1993). Though the helical repeats determined from S®I reactions on supercoiled and linear DNA carried overlapping error margins, the imposition of the value from linear DNA (10.9 bp/turn) onto the data from the supercoiled substrates (Figure 5a), or the value from supercoiled DNA (11.5 bp/turn) onto the data from the linear substrates (Figure 8a), both resulted in larger sum-of-squares deviations than those obtained with the optimal ®ts (analyses not shown). It thus seems likely that the increase in the helical repeat from the standard value of 10.5 bp/turn to that of 10.9 bp/turn, seen with S®I on linear DNA, is due primarily to the raised temperature while the further increase from 10.9 to 11.5 bp/turn, seen with S®I on intact plasmids, is a consequence of the linking de®ciency of negatively supercoiled DNA.

Conclusions formation of products with single-strand breaks, which arise whenever the looping interaction falls apart before either site suffers a double-strand break. In contrast, the reactions on linear substrates were assessed from the fraction of the DNA converted directly to the ®nal product cut in both strands at both sites, a process that requires the loop to remain extant until four phosphodiester bonds have been cleaved. The data in Figure 8 thus provide an indication of the relative stability of the looped complex while those in Figure 5 indicate the converse, loop instability.

The S®I restriction endonuclease has to interact with two copies of its recognition sequence before it can cleave DNA (Wentzell et al., 1995; Nobbs & Halford, 1995). Previous studies has eliminated the possibility that this type II enzyme mediates its interaction with two sites by tracking along the DNA from one site to the other (Szczelkun & Halford, 1996), in the manner of the type I and the type III restriction enzymes (Meisel et al., 1995; Szczelkun et al., 1997). The data presented here demonstrate that the interaction with two S®I sites in cis involves the formation of a DNA loop between the sites, presumably as a result of the tetrameric protein binding simultaneously to both

442 sites. A characteristic property of a looping interaction is its cyclic dependence on the length of DNA between the two sites (Schleif, 1992). The reactions of the S®I endonuclease on either supercoiled or linear DNA carrying closely spaced recognition sites matched this expectation for periodic variations, not necessarily in terms of the turnover rate of the enzyme (Figure 3) but rather in the nature of the products liberated from the enzyme at the end of each turnover (Figure 5 and 8). The turnover rate of an enzyme acting at two DNA sites will be affected by the separation of the sites only if the looping interaction impinges on the rate-limiting step(s) of the reaction. On linear DNA with two S®I sites, the way in which the turnover rates varied with site separation was the same as that for the partitioning of the substrates to doubly cut products. The linear substrates on which S®I had particularly low turnover rates, such as those with inter-site spacings of 149, 158 and 170 bp (Figure 3b), all gave comparatively low yields of doubly cut product (Figure 8), and vice versa. Hence, on linear DNA, the looping interaction affects in parallel the rate of DNA cleavage and the distribution between fully and partially cleaved products. Indeed, the linear DNA with S®I sites 162 bp apart gave an unexpectedly high yield of doubly cut product, relative to those with spacings of 160 or 164 bp, yet this DNA also gave an unexpectedly high reaction rate; for some unknown reason, this particular substrate is peculiarly adept at loop formation. However, the supercoiled substrates invariably gave faster turnover rates than the same DNA in linear form and their reaction velocities showed no systematic variation with altered spacings (Figure 3a). Even so, the sinusoidal variations in the yield of open-circle DNA from the reactions on the intact plasmids provide evidence for looping between S®I sites on supercoiled DNA (Figure 5). Nevertheless, on supercoiled DNA, the formation of the loop has no impact on the rate-limiting step(s) in the reaction pathway. The binding of a protein to two DNA sites requires the intervening DNA to be bent into a curve that spans the two DNA-binding surfaces on the protein (Lewis et al., 1996). Consequently, as the distance between the sites is progressively reduced, the formation of the loop should be progressively disfavoured, leading eventually to the state when the loop can no longer be formed because the free energy needed to bend the DNA will outweigh that gained from the binding of the protein to both sites. For example, the looping interaction between the Hin recombinase and its accessory protein FIS needs a minimum of 105 bp, though this is reduced to 60 bp in the presence of the DNA-bending protein HU (Haykinson & Johnson, 1993). Other systems form loops across <60 bp of DNA (Lee & Schleif, 1989). In this study, with inter-site spacings from 104 to 1023 bp, no minimal distance was found that was too short to allow S®I to form a loop. If such a

Looping by S®I

minimum had been found, it would have resulted in the same DNA cleavage rates as in the reactions on substrates with one S®I site, but all of the substrates with two sites were cleaved more rapidly than those with one site (Figure 2). However, while the linear substrates retained the cyclic response between partially and fully cleaved products as the distance between the S®I sites was reduced from 113 to 104 bp (Figure 8b), the supercoiled substrates behaved differently with these short spacings and gave progressively more of the partially cleaved product (Figure 5b). The reason for this difference may be that the requisite bending of the DNA has to be imposed, in the case of supercoiled DNA, onto a molecule whose helical axis is already curved, from supercoiling itself. Nonetheless, two S®I sites 104 bp apart, the minimal tested here, were attacked more rapidly on supercoiled DNA than on linear DNA (Figure 3). This provides a direct illustration of the role of supercoiling in facilitating communications between DNA sites in cis.

Materials and Methods To construct the plasmids pW104 and pW211 (Figure 1), pGB1 was cleaved with MluI and then treated with exonuclease III for various times, followed successively by nuclease S1, Klenow polymerase and bacteriophage T4 DNA ligase (Promega ``Erase-a-Base'' kit). The ligated DNA was used to transform E. coli HB101 (Sambrook et al., 1989). One transformant was found to contain a derivative of pGB1 with 104 bp between the S®I sites (named pW104) and another with 211 bp (named pW211). Further manipulations of pW211, by ExoIII/S1 (Henikoff, 1987) starting from its SpeI site (Figure 1) and by additional methods from Sambrook et al. (1989), yielded the other plasmids in the pW104 to pW211 series (Wentzell, 1997). For each plasmid, the sequence of the DNA between the two S®I sites was determined in full (International Blood Reference laboratory, Southmead Hospital, Bristol), in order to identify the precise number of bp separating the sites. Transformants of E. coli HB101 carrying each plasmid were grown in M9 minimal medium with 1 mCi/litre [methyl-3H]thymidine. The covalently closed form of the DNA was puri®ed by density gradient centrifugations in CsC1/ethidium bromide (Nobbs & Halford, 1995). All other materials and methods were as described (Wentzell et al., 1995; Vipond et al., 1995; Nobbs et al., 1998a), except that all S®I reactions in this study were carried out in HS (high salt) buffer. HS buffer is 10 mM Tris-HCl (pH 7.5), 200 mM NaCl, 10 mM MgCl2, 5 mM b-mercaptoethanol and 100 mg/ml bovine serum albumin.

Acknowledgements We thank Symon Erskine, Niall Gormley, Tony Maxwell, Susan Milsom, Mark Szczelkun and Mark Watson for aid and advice, and Ira Schildkraut (New England Biolabs) for the S®I system. This work was funded by the BBSRC and the Wellcome Trust.

Looping by S®I

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Edited by J. Karn (Received 3 March 1998; received in revised form 26 May 1998; accepted 4 June 1998)