DNA translocation by the restriction enzyme from E. coli K

Cell, Vol. 20, 237-244,

May 1980,

Copyright

0 1980

DNA Translocation E. coli K

by MIT

by the Restriction

Robert Yuan, Daniel L. Hamilton and Jean Burckhardt Cancer Biology Program National Cancer Institute Frederick Cancer Research Center P.O. Box B Frederick, Maryland 21701

Summary The restriction endonuclease Eco K binds to a host specificity site and then proceeds to cleave the DNA at sites that may be several thousand bases away. It does this by translocating the DNA past the enzyme in an ATP-dependent reaction that results in the formation of highly twisted loop intermediates. DNA cleavage can occur on either side of the host specificity site. Introduction In recent years, restriction endonucleases have become an essential tool in genetic, structural and functional studies of DNA. The use of these enzymes in the construction of new genomes has attracted widespread attention and considerable debate. What has been less evident is that these enzymes provide an excellent system for the study of protein-DNA interactions. An interesting question that can be approached by looking at such systems is that of DNA translocation. This problem centers around the mechanism by which a protein binds at a specific DNA site and yet acts at sites that may be thousands of bases away. Restriction endonucleases have been purified from a wide variety of bacterial strains. A bacterial strain will produce a restriction enzyme that is specific for DNA replicated in other strains. At the same time, it will have a DNA methylase that will protect its own DNA from the action of its own restriction enzyme. These endonucleases have been classified into two groups (Boyer, 1971). Type II enzymes such as Eco RI appear to be simple proteins that require only magnesium ions for their endonucleolytic activity. [The nomenclature used for these enzymes is the one proposed by Smith and Nathans (1973).] DNA methylation is catalyzed by a separate enzyme that utilizes Sadenosylmethionine (AdoMet) as the methyl donor. Type I enzymes such as Eco K and Eco B are complex proteins that catalyze three different reactions. They cleave unmodified DNA in the presence of AdoMet, ATP and Mg++ (Linn and Arber, 1968; Meselson and Yuan, 1968). The restriction reaction is accompanied by an ATP-hydrolyzing activity that continues after DNA cleavage has gone to completion (Yuan, Heywood and Meselson, 1972). The same enzymes also

Enzyme from

act as DNA-methyl transferases that modify the genetically mapped host specificity sites on the DNA (Haberman, Heywood and Meselson, 1972). It is these DNA sites that confer susceptibility to a given restriction and modification system. The reaction mechanism of the restriction endonuclease from E. coli K (called Eco K) has certain features that are important in addition to the specialized study of restriction and modification. One of these, recognition of nucleotide sequences, is common to most proteins that specifically interact with DNA. A less common characteristic is the way in which recognition of the nucleotide sequence modulates the enzyme into one of three modes of action: restriction, modification, or release of the protein from the DNA. Perhaps its most unusual feature is that, whereas DNA methylation occurs at the host specificity sites (Smith, Arber and Kuhnlein, 1972) DNA cleavage occurs at a large number of sites that are distinct from them (Horiuchi and Zinder, 1972). How does Eco K specifically bind to a host specificity site (SK site) and then proceed to cleave the DNA at sites that may be several thousand bases away? Four different mechanisms have been proposed. All four require that the activated enzyme must first interact with an unmodified SK site. In the random walk model, Eco K would dissociate from the SK site and be able to interact at random sites on the same or a different DNA molecule. This is highly improbable, since the enzyme is unable to cleave modified DNA in trans when incubated with a mixture of modified and unmodified DNA (Meselson and Yuan, 1968). The directional walk model would have ATP trigger the movement of the enzyme (or subunits of it) along the DNA molecule until it reached one of the possible cleavage sites. The evidence to date indicates that the enzyme remains at the recognition site even following endonucleolytic action (Bickle, Brack and Yuan, 1978). The random flop model would have Eco K stably bound at a SK site. Cleavage would result from a random second contact of the same DNA molecule with the enzyme. The DNA winding model proposes that Eco K remains at the SK site and winds the DNA past it until it comes into contact with one of the possible cleavage sites. In this paper, we present evidence for a mechanism of DNA translocation which incorporates elements of the latter two models. Results Steps in DNA Recognition by Eco K Eco K binds AdoMet and forms an activated enzyme (Eco K*) (Hadi, Bickle and Yuan, 1975). Eco K* forms an initial complex with any DNA regardless of the presence or absence of SK sites. The SK sites can exist in three possible forms: modified (methylated on both strands), unmodified (unmethylated on both

Cell 238

strands), and heteroduplex (one strand methylated and one unmethylated). Eco K* binds tightly to all three of these SK sites to form recognition complexes. ATP then induces a conformational change in the enzyme that allows it to discriminate between these different forms of the SK site (Bickle et al., 1978). In the case of modified sites, Eco K* is released from the DNA, while with heteroduplex sites, the enzyme remains in situ and methylates the unmodified strand. With unmodified sites, the recognition complex is first transformed into a new complex that is characterized by its retention on nitrocellulose filters. Electron microscopic examination of this complex showed that a major conformational Eco K* had undergone change, but did not distinguish between the loss of a subunit and an allosteric effect. This altered form of the enzyme was called Eco K+. The same effect could be obtained with the P,y-imido analog of ATP, indicating that ATP hydrolysis was not required for the preceding reactions. DNA scission, which is dependent on ATP hydrolysis, then occurs at cleavage sites that are distant from the SK sites. Mapping of Eco K on pBR322 DNA The continuation of our studies into the reaction mechanism of Eco K required a DNA substrate of low molecular weight for which sequence data were available. The DNA of the plasmid pBR322, which has 4362 bp, contains two SK sites, and is readily cleaved by Eco K, was ideally suited for our purposes. Both the nucleotide sequences of the SK sites (Kan et al., 1979) and pBR322 DNA (Sutcliffe, 1978) had been determined, and the SK sites had been located at positions 1663-1651 and 4024-4033. The SK sequence was 5’-A A C-6 bases-G T G C-3’, a hyphenated structure with two constant domains of three and four bases, respectively, separated by a spacer of six bases. The asymmetric nature of the sequence results in the two SK sites being oriented in opposite directions on pBR322 DNA. Eco K molecules could be localized on unmodified pBR322 DNA by electron microscopy. Linear pBR322 DNA was prepared by digestion with Eco RI, Barn HI or Pst I. Recognition complexes were formed by incubation of these DNA substrates with Eco K in the presence of AdoMet and adenosine-5’-/3,y-imidotriphosphate, followed by glutaraldehyde fixation. The imido analog of ATP stabilizes the specific binding without any DNA cleavage, while eliminating nonspecific interactions between Eco K and DNA. The complexes were then prepared for electron microscopy by the protein-free method (Brack et al., 1976). Examination of the electron micrographs showed that most DNA molecules had two enzyme molecules bound to them. The positions of these enzyme molecules on Barn HI-generated linear DNA (Figure l), taken in conjunction with the results with Eco RI and Pst I linears, localized the enzyme molecules at posi-

tions 1660 and 4020 (& 50 bases) (Figure 2). This agrees with the positions predicted from the DNA sequencing data. Recognition complexes prepared with unmodified pBR322 supercoiled DNA also showed two enzymes per DNA molecule separated by the expected distances (data not shown). Intermediates in DNA Translocation Having once ascertained that Eco K* binds to the two SK sites, we proceeded to look for possible intermediates during DNA cleavage. pBR322 DNA was used as the DNA substrate in one of three possible forms: linear molecules and supercoiled or relaxed covalent circles. The complexes between Eco K* and supercoiled unmodified pBR322 DNA were the first ones to be studied. Before ATP addition, Eco K* is bound to the SK sites in a recognition complex (Figure 3A). Incubation with ATP yielded linear molecules and DNA fragments with Eco K+ still bound to the SK sites (Figure 38). We tried to synchronize the reaction by beginning with recognition complexes, which had been incubated with heparin, in order to eliminate nonspecific enzyme-DNA complexes (Bickle et al., 1978). ATP was added to trigger DNA cleavage. Aliquots were removed at 10 set intervals, fixed immediately with glutaraldehyde, and prepared for electron microscopy by the protein-free method. Several novel structures were observed: -Regular loop: supercoiled DNA with Eco K+ making a two-point attachment to form a loop (Figure 4A). -Twisted loop: supercoiled or relaxed DNA with a tightly wound loop that has its origin at an Eco K+ molecule (Figures 4B and 4C). -Double-twisted loop: this structure is similar to the twisted loop, but appears to be generated by two enzyme molecules translocating DNA towards each

6

10

20

30

40

50

60

70

60

90

100

Fractional Length Figure 1. Histogram of Eco K Bound Generated by Barn HI Digestion

to pBR322

Linear

Molecules

pBR322 DNA was digested with Barn HI under standard conditions. The linear molecules were phenol-extracted and precipitated with ethanol. Recognition complexes of Eco K and pBR322 DNA were formed by incubation in the presence of AdoMet and adenosine-5’P,y-imido-triphosphate (Bickle et al., 19781, fixed in 0.1% glutaraldehyde, and mounted for electron microscopy as described in Experimental Procedures. Enzyme position relative to DNA length was measured with a Numonics Graphic Calculator. The positions of the two SK sites were confirmed by mapping similar complexes with linear pBR322 DNA generated by digestion with Eco RI and Pst I.

Mechanisms 239

of DNA Restriction

EcoR

I

Barn HI (375)

PV; II (2067) Figure 2. Map of the SK Sites in Relation Selected Type II Restriction Enzymes

structures. The result with the imido analog was of particular interest, since it induced the same conformational change from Eco K* to Eco K+ as ATP, but did not allow DNA cleavage. dATP does allow endonucleolytic activity, though at reduced efficiency. This limited reaction is reflected in the formation of the intermediates. This series of experiments allowed us to conclude that the appearance of loop structures is associated with DNA cleavage and is dependent on ATP hydrolysis. Eco PI5 is a member of a family of restriction enzymes that show certain similarities to the Type I enzymes-that is, they are large proteins that can both cleave and methylate unmodified DNA (Reiser

(1662)

to the Cleavage

Sites of

The numbers under each site represent the beginning of the given nucleotide sequence using the Eco RI site as the origin. The cleavage sites for the Type II enzymes were taken from Sutcliffe (1978). The arrows at the SK sites represent the orientation of the sequence as A A C-6 bases-G T G C.

other (Figure 4D). These intermediates are much less frequent than the others. We also analyzed the electron micrographs to determine the kinetics of formation of these new complexes (Figure 5). The results are consistent with the following sequence of events: recognition complexes + regular loops + twisted loops -+ linears and fragments. Similar results were obtained when heparin was omitted from the reaction. Starting with a population of 80-95% recognition complexes, we consistently observed the appearance of a maximum of 1 O25% of the total DNA molecules as regular loops and 5-15% as twisted loops. These results suggest that these new complexes are intermediates in the endonuclease reaction which bring the cleavage sites into contact with the enzyme bound at the SK site. The characterization of these loop structures as intermediates in the cleavage reaction was supported by a series of control experiments. In the absence of ATP, no loop structures were observed. An aged preparation of Eco K, which formed recognition complexes but did not cleave DNA, was used. No loop structures were detected. If modified pBR322 DNA was used as the substrate, addition of ATP resulted in the release of Eco K* from the recognition complexes, as expected from previous results (Bickle et al., 1978). A number of ATP analogs were used in place of ATP. Three of them, adenosine-5’-a&methylene-triphosphate, adenosine-5’-/3,y-imido-triphosphate, and adenosine-5’-/3,y-thio-triphosphate, do not sustain DNA cleavage, nor do they generate loop

Figure 3. Electron Cleavage Products

Micrographs

of

Recognition

Complexes

and

(A) Recognition complexes. (8) Cleavage products resulting from incubation of recognition complexes with ATP for 5 min at 3O’C. The recognition complexes were formed by incubation of unmodified pBR322 DNA with Eco K in the presence of AdoMet and were prepared for electron microscopy as described in the legend to Figure i.Bar=lOOnm.

Figure

4.

Electron

Micrographs

of Cleavage

Intermediates

These complexes were observed in the process of DNA cleavage: (A) regular loop; (B and C) twisted loops at different stages; (D) a doubletwisted loop. Recognition complexes were prepared as described in the legend to Figure 3. DNA cleavage was initiated by the addition of ATP, and samples were removed at time intervals of 0. 5, 15, 25. 35, 45, 60 and 300 sec. They were prepared for electron microscopy as indicated in the legend to Figure 1. Bar = 100 nm.

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100 90 80

Figure 6. Electron ear Lambda DNA

Time Figure

5. Kinetics

of Cleavage

(set)

Micrograph

of Twisted

Loop Intermediate

on Lin-

Cleavage intermediates were prepared as described in the legend to Figure 4 except that linear lambda DNA was used as the substrate. The highest proportion of these intermediates appeared 35 set after ATP addition. Bar = 100 nm.

Intermediates

Electron micrographs were prepared from the same time samples usad in Figure 4. Approximately 300 molecules from each time point were scored as: (u) recognition complexes; (M) relaxed looPs; (A-A) twisted iOOPS; w) linears plus fragments with enzyme.

and Yuan, 1977). The endonuclease reaction requires ATP (and is stimulated by AdoMet); however, no extensive ATP hydrolysis is observed. Unlike Eco K, this enzyme cleaves the DNA at sites quite near the recognition site (25-26 bases in the 3’ direction), with no apparent indication of sequence specificity at the site of scission. Experiments similar to the ones described for Eco K have been carried out with Eco Pi 5, and no regular or twisted loop intermediates were detected, even though DNA cleavage was efficient. This is consistent with the idea that ATP hydrolysis is required for DNA translocation over large distances. A number of attempts have been made to increase the proportion of loop structures by manipulating such variables as temperature and the concentration of ATP and Mg++. Mg++ has also been replaced with Mn++, Co+‘, Ni++, Ca++ and Zn++. In general, it was found that the rate of intermediate formation was either slowed down or abolished altogether, with a corresponding effect on DNA cleavage. A different approach involved the quantitative conversion of the Eco K* in the recognition complex to Eco K+ by incubation with the imido analog of ATP. We had expected that the addition of an excess of ATP to these filter-binding complexes would lead to the synchronized appearance of large numbers of loop intermediates. No increase in the rate or yield of loop structures was observed. As will be discussed below, the proportion of intermediates observed probably reflects features of the enzyme mechanism.

Experiments similar to those described for supercoiled DNA were repeated with relaxed and linear pBR322 DNA. Relaxed circular pBR322 DNA was cut at approximately the same rate as the supercoiled form. Both regular and twisted loop complexes were observed, and their kinetics of formation were comparable to those with supercoils. Linear molecules of pBR322 (generated by Eco RI or Barn HI digestion) were efficiently cleaved by Eco K. However, several attempts to show DNA translocation were unsuccessful. The most likely explanation is that the topology of a circular DNA imposes certain constraints on the rate of DNA translocation as more and more DNA is wound past the enzyme. In the case of pBR322 linear molecules, the DNA could be rapidly wound off the end in the absence of such constraints. If this is indeed the case, a longer linear substrate might allow detection of the loop intermediates. Indeed, the intermediate complexes were observed with unmodified linear lambda DNA (Figure 6). The characterization of loop structures provides a solution to the problem of how Eco K+ can remain bound to its SK site and still cleave at sites thousands of bases away. If the SK site is unmodified, Eco K+ translocates the DNA past it in a manner that generates a highly supercoiled loop structure. This DNA translocation is an ATP-dependent reaction that is terminated by cleavage at one of many potential sites. Direction of DNA Translocation As we have seen in a previous section, the sequence of the SK site is asymmetric. Current dogma dictates that an enzyme would bind to such a sequence in only one orientation, thus directing translocation and ultimately DNA cleavage on only one side of the SK site.

Mechanisms 241

of DNA Restriction

pJW

401

3907 bp

SKI 7

Ava I

80

3666

SKI f

III

2373 SKI f

Pvu II

404

3503 7. A Map of pJW401

The numbers a terminus.

A

represent

B

DNA Linear

the number

C

I

P 7Ot

1634

Figure

3 60 ijjl;:c:-::--90

239

Hind

100

Molecules

of bases between

D

an SK site and

E

5

F

15

10 Time

Figure

9. Kinetics

of ATP Hydrolysis

20

25

30

(min) with pJW401

Linear

Molecules

Full-length linears of pJW401 were generated by digestion with either Ava I, Hind Ill or Pvu II. These DNA substrates were used in the restriction reaction with ‘H-ATP; aliquots were removed at the indicated time intervals, and ATP hydrolysis was measured by a standard assay. (Yuan et al., 1972). (U) pJW401 linears from Ava I; (-1 pJW401 linears from Pvu II; (A-A) pJW401 linears from Hind Ill.

Figure

8. Cleavage

of pJW401

DNA Molecules

by Eco K

Plasmid pJW401 DNA was linearized with either Ava I, Hind Ill or Pvu II. The DNA was purified, incubated with 9 yl of a solution of Eco K for 1 hr at 3O’C and electrophoresed on an agarose gel as described in Experimental Procedures. (B) and (E) contained 0.4 pg of DNA, while 0.5 pg of DNA was used for (A), (C), (D) and (F). (A) is Ava I linear DNA before Eco K digestion, (D), after digestion: (8) is Hind Ill linear DNA before digestion, (E), after digestion: and (C) is Pvu II linear DNA before digestion, (F), after digestion.

To test the hypothesis of asymmetric translocation and cleavage by Eco K, we utilized a substrate containing a single SK site. pJW401 is a derivative of pBR322, which has had deleted the region of 452 bp (carrying the sK2 site) between the Hind II and the

Eco RI sites (Figure 2). The supercoiled form of pJW401 was cleaved efficiently by Eco K, but at a rate approximately 3 times slower than pBR322. Utilizing the circular form of pJW401, regular and twisted loop intermediates were observed by electron microscopy at frequencies similar to pBR322 (data not shown). To test whether the orientation of the SK site affected cleavage, we prepared several linear forms of pJW401 using various Type II restriction enzymes which cut this genome only once. Ava I cuts pJW401 239 bases from one side of the SK site, Pvu II cuts 404 bases to the other side, and Hind III cuts 1634 bases away (Figure 7). The Hind Ill linear is a molecule with the SK site located approximately in the middle; it was used as a control to determine the reaction conditions for Eco K cleavage. If the orientation of the SK site determines the direction of cleavage, we would expect that the Ava I linear would be cleaved, whereas the Pvu II linear would be resistant, or vice versa. All three linears were incubated with Eco K, and the digests were analyzed by agarose gel electrophoresis (Figure 8). DNA cleavage was approximately the same in all three cases. These results were confirmed by monitoring the restriction reaction by a more sensitive but indirect reaction, which consists of following the ATP hydrolysis that accompanies the endonuclease activity (Figure 9). As seen in Figure 9, all three linears generate ATP hydrolysis. The rates of ATP hydrolysis vary, however, with the highest values for the Ava I linears, followed by Pvu II linears, and with the lowest values in the case of Hind Ill linears. The results of these experiments strongly suggest that Eco K is capable of translocating and cleaving the DNA on

Cell 242

ATP ADP + Pi

Figure

10.

Model

of the DNA Translocation

Mechanism

by Eco K

Eco K* binds at an unmodified SK site and forms a filter binding complex in the presence of ATP due to a conformational alteration of the enzyme, shown as Eco K+ on the second line of the Figure. This altered complex can now bind at a second site on the DNA substrate, presumably in any of the four configurations depicted on the first and third lines. (A and B are used to indicate relative ends of the DNA molecule; the arrows depict the direction in which translocation will occur.) Concurrent with ATP hydrolysis, the Eco K winds the DNA past itself, and, depending upon the orientation of the second binding site, it will either form a twisted loop which will increase in size (left side of fourth line) or decrease in size (right side of fourth line).

either side of the SK site. Given the ATPase is possible that there is some preference cleavage in one direction.

results, it for DNA

Discussion The restriction endonuclease from E. coli K is activated by AdoMet and binds specifically to SK sites in any of its three possible configurations (modified, unmodified and heteroduplex). The process by which Eco K cleaves at sites several thousand base pairs away requires ATP in two distinct steps. ATP acts first as an allosteric effector that converts Eco K* into a new form, Eco K+ that can now discriminate between methylated and unmethylated SK sites. Eco K+ will dissociate from methylated sites but remain bound at unmethylated ones. The same effect is observed with the imido analog of ATP (Bickle et al., 1978), indicating that ATP hydrolysis is not required for this step. In order for Eco K+ to cleave the DNA at sites distal to the SK sites, it translocates the DNA past it by a mechanism that is coupled to ATP hydrolysis. The experiments described here, together with studies on DNA gyrase (Liu and Wang, 1978; Gellert

et al., 1978; Peebles et al., 19781, have led us to propose a model for DNA translocation by Eco K which would consist of the following steps. Two-Point Contact Eco K* would have two DNA-binding sites, one of which is specific for the SK sequence and binds to it in a particular orientation. The second DNA-binding site is not sequence-specific, and does not become available until ATP has induced the conformational change to Eco K+ (presumably by the loss of one or more subunits). The DNA on one side of a SK site can diffuse back in either of two possible orientations and make contact with this nonspecific binding site on Eco K+. The cleavage experiments with the pJW401 linear DNAs are consistent with the second contact being possible on either side of the SK site. A regular loop can therefore have four possible configurations (see Figure 101, its size being limited by the ability of the DNA helix to bend back on itself. The formation of regular loops would be readily reversible in the absence of ATP hydrolysis, as suggested by the fact that they were present only after ATP addition. The random character of this first step would explain our inability to increase the degree of synchrony in generating loop intermediates. It should be noted that although most recognition complexes on pBR322 DNA have two enzyme molecules bound to them (one at each SK site), very few of the twisted loop structures have two enzyme molecules winding DNA simultaneously. We presume this to mean that once an enzyme has, begun to translocate DNA past it, it makes it difficult for the rest of the supercoiled DNA to fold back to make contact with the second enzyme. Furthermore, the probability of both enzymes making a second point contact at the same time is very low. DNA Winding While bound to the SK site, Eco K+ begins to wind the DNA past the second DNA-binding site in an ATPdependent reaction. Regardless of the original configuration of the regular loop, the direction of winding will always be the same. The process of DNA translocation results in the formation of twisted loops with a helical density which is much higher than that of the original pBR322 or PM2 supercoiled DNA. It is not known whether these structures are overwound or underwound. Three of the mechanisms proposed for DNA gyrase (Liu and Wang, 1978; Gellert et al., 1978; Peebles et al., 1978) can also be used to explain this aspect of the Eco K reaction. In one mechanism, the enzyme tracks along the major (or minor) groove of the DNA helix, thus rotating it as it moves past. Another possibility is one in which the moving DNA would not rotate, but Eco K+ and the SK portion of the DNA to which it is bound are rotated by the passage of the DNA. A third mechanism is one in which a negative superhelical turn is introduced by wrapping the DNA around the enzyme a full turn. Though our results do

Mechanisms 243

of DNA Restriction

not prove any of these mechanisms, we have a preference for the first one, since it would allow the enzyme to continuously monitor the same side of the DNA helix until it encountered those sequence and/ or structural features that characterize a cleavage site. One problem that we have encountered in analyzing our results with circular DNA (supercoiled or relaxed) is that if the enzyme introduces negative turns into the downstream loop, an equivalent number of positive turns would be generated in the other portion of the molecule. One would therefore expect to detect molecules with twisted loops on both sides of the enzyme. In fact, the upstream portion of twisted loop structures shows either a relaxed structure or appears to have the same degree of superhelicity as the original material. The easiest way around this dilemma is to assume a nicking-closing activity that acts on the upstream loop. At present, there is no evidence for such an activity. To examine whether Eco K and DNA gyrase have similar steps in their reaction mechanism, we have tested the effect of novobiocin and nalidixic acid on Eco K. Both of these drugs are good inhibitors of DNA gyrase (Gellert et al., 19761, but have no effect on either the formation of filter-binding complexes or DNA cleavage by Eco K. Electron microscopic studies of Eco B, an enzyme similar to Eco K, have been reported recently by Rosamond, Endlich and Linn (1979). Loops of duplex DNA were detected, usually at or near the termini, following incubation with the enzyme. No supertwisted loops were observed. These results, along with cleavage experiments, were used to postulate a model for unidirectional “tracking” and cleavage of DNA. The difference between these results and ours can be explained in terms of the design and execution of the experiments. All of our reactions were done starting with recognition complexes (an intermediate), adding ATP to initiate a synchronized start of the cleavage process, and taking samples from O-60 sec. In contrast, Rosamond et al. (1979) added enzyme to a complete reaction mixture and carried out incubations of 3-5 min, by which time the supercoiled fd DNA had been cleaved one or more times. They were therefore working primarily with the products rather than the intermediates of the cleavage reaction. As we have shown, small linear DNA does not lend itself to the characterization of twisted loop structures. The exciting developments with DNA gyrase and its involvement in DNA replication and recombination represented a new and unusual form of enzymatic activity. Our work on Eco K may indicate that mechanisms by which a protein binds to specific sequences and translocates DNA past it may be less uncommon than one would think. DNA translocation provides an alternative to movement along the DNA, and it might not necessarily disturb other proteins bound to the

DNA in the in vivo situation. The existence of twisted loop structures has an interesting implication. Sitespecific recombination (Nash et al., 1977) and transcription of certain promoters (Puga and Tessman, 1973; Schuman and Schwartz, 1975; Falco, Zivin and Rothman-Denes, 1978) have been shown to be dependent on DNA supercoiling. Mechanisms such as those described here, which generate transient, highly supercoiled DNA structures, might represent a new form of regulatory control. It is certain that we can expect to find other examples of DNA translocation in the near future. Experimental

Procedures

Preparation of Enzymes and DNA Substrates Endonuclease Eco K was purified to homogeneity as described by Buhler and Yuan (1978) for the preparation of the mutant enzyme Eco K-i 8, with the following modifications: the DNA-agarose column was replaced by a heparin-sepharose column prepared according to the method of Bickle, Pirrotta and lmber (1977); and the presence of enzyme activity was followed during its purification by assaying cleavage of unmodified versus modified pBR322 DNA on agarose gels or by measuring the filter binding activity (Meselson and Yuan, 1971). The purity of the enzyme fraction was monitored by electron microscopy, and the protein concentration was determined according to the method of Lowry et al. (1951 I Pure enzyme fractions containing 0.64 pg/ml of protein were stored in 20% glycerol, 20 mM potassium phosphate (pH 7.5), 0.1 mM EDTA and 7 mM mercaptoethanol at -2ooc. The plasmids, pBA322 and pJW401, were prepared from either r-mor r+m+ E. coli cells after amplification of the plasmid by chloramphenicol as described by Clevell (1972). pJW401 is a derivative of pBR322 constructed in the laboratory of J. Wang, and has had one of the two SK sites deleted (see Figure 2). It was given to us by J. Wang for use in these experiments. Phage h DNA was obtained by thermal induction of ah cl857susS7 prophage present in either a rrkmmk or r+kmtk strain. Endonuclease Assay Digestion of the DNA with Eco K was performed at 30°C for 1 hr in 100 pl reaction assays containing 25 mM HEPES (pH 8.0). 6 mM MgCl*, 12 mM mercaptoethanol, 0.26 mM EDTA, 0.5 pg DNA, 0.5 PM ATP, 66 mM AdoMet, and different concentrations of Eco K. The incubation mixtures were phenol-extracted and concentrated with isobutanol to 34 pl before the DNA was analyzed on a 1.2% agarose gel. The gel was stained afler electrophoresis for 30 min in a 1 pg/ml ethidium bromide solution, washed with water for 10 min, and photographed using a short wavelength ultraviolet transilluminator. ATPase Assay The ATPase assay Yuan (1971).

was

performed

as described

by Meselson

and

Electron Microscopy Samples for electron microscopy were prepared as follows. DNA was added to a concentration of approximately 1 pg/ml to a buffer containing 0.1 mM HEPES (pH 8.0), 1.3 mM EDTA, 6 mM MgS04 and 12 mM mercaptoethanol. To this solution, sufficient Eco K was added so that 90% or more of the DNA molecules had one or more enzymes bound to them. Addition of ATP and time of incubation are described in the text. After the reaction had been stopped by the addition of an equal volume of 0.2 M glutaraldehyde, the DNA was spread by the protein-free method using carbon grids glow-discharged in a pentylamine atmosphere @rack et al., 1976). After spreading, the grids were stained briefly with uranylacetate.

Cell 244

shadowed at an angle of 8’ with Pt:Pd (80:20) and examined in a Hitachi HU 12-A electron microscope. Negatives were viewed on a microfiche reader, and measurements were made with a Numonics Electronic Graphics Calculator. Acknowledgments We are grateful to Drs. Martin Gellert. Kyoshi Mizuuchi, Richard Musso and Stuart Austin for helpful criticism of the manuscript. We would also like to thank Dr. Christine Brack for her contribution in setting up the electron microscopy techniques, and Dr. James C. Wang for his help with certain experiments and for many thoughtful discussions during the course of this project. These experiments benefited from the excellent technical assistance of Phuong-Van T. Luc and Jane Weisemann. J. B. is a recipient of the Swiss National Foundation Fellowship. This research was sponsored by the National Cancer Institute under contract with Litton Bionetics, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

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in

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Gellert, M., Mizuuchi, K., O’Dea, M. H., Ohmori, H. and Tomizawa, J. (1978). DNA gyrase and DNA supercoiling. Cold Spring Harbor Symp. Quant. Biol. 43, 35-40. Haberman, A., Heywood J. and Meselson, M. (1972). DNA modification methylase activity of Escherichia coli restriction endonucleases K and P. Proc. Nat. Acad. Sci. USA 69, 3138-3141. Hadi, S. M., Bickle, T. A. and Yuan, R. (1975). The role of Sadenosylmethionine in the cleavage of deoxyribonucleic acid by the restriction endonuclease from Escherichia coli K. J. Biol. Chem. 250, 4159-4164. Horiuchi, K. and Zinder, N. D. (1972). Cleavage of bacteriophage fl DNA by the restriction enzyme of Escherichia coli B. Proc. Nat. Acad. Sci. USA 69, 3220-3224.

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