Steps in the reaction mechanism of the Haemophilus influenzae Rf restriction endonuclease

J. Mol. Biol. (1982) 154, 615-627

Steps in the Reaction Mechanism of the Haemophilus influenzae Rf Restriction Endonuclease RYSZARD BRZEZIJISKI AND A N D R Z E J PIKKABOWICZ

Institute

of Microbiology, Warsaw University 00-046 Warsaw, Poland

(Received 14 April 1981, and in revised form 30 October 1981)

Hinflll is a restriction modification enzyme isolated from Haemophilus influenzae strain Rf. It requires ATP for cleavage and iS-adenosyl-L-methionine for methylation of DNA. jS-Adenosyl-L-methionine acts as an allosteric effector in the endonuclease reaction. The enzyme forms a complex with unmodified DNA in the absence of ATP and iS-adenosyl-L-methionine. This complex is sensitive to inhibition by heparin. S-Adeiiosyl-L-methionine is required for the formation of a complex that is insensitive to such inhibition. ATP acts as an allosteric effector of HinflU, and induces DNA cleavage followed probably by the release of the enzyme from the DNA.

1. Introduction Restriction endonucleases of t y p e III have emerged from the purification a n d characterization of the enzymes coded by the closely related bacteriophage P I a n d plasmid P 1 5 (Habermanj 1974: Reiser & Y u a n , 1977), as well as b y t h e e n z y m e isolated from Haem^ophilus influenzae R f (Piekarowicz et al., 1974; K a u c & Piekarowiez, 1978). They are complex proteins with two different subunits (T. A. Bickle, unpublished results), a n d can catalyse b o t h D N A cleavage a n d modification methylation. They require A T P and magnesium ions for D N A cleavage, a n d t h e restriction activity is stimulated b y AdoMetf. Digestion of unmodified D N A with these enzymes produces distinct fragments, even t h o u g h D N A digestion is n o t complete; i.e. scission does n o t occur a t every possible cleavage site. I n t h e presence of AdoMet alone, all these enzymes are able t o m e t h y l a t e the unmodified D N A , protecting it against cleavage. When A T P is present, AdoMet plays a dual role: it stimulates the restricting activity of t h e enzymes, a n d acts as a d o n o r of methyl groups for methylation. Therefore, these enzymes can modify a n d cleave D N A simultaneously. T h e restriction endonucleases EcoPl a n d EcoFlS recognize a short nucleotide t Abbreviations used: imidotriphosphate.

AdoMet,

S-adenosyl-L-methionine;

imido-ATP,

adenosine-5'-{/3, y)-

615 0022-2836/82/040615-13 .W2.00/0 23

© 1982 Academic Press Inc. (London) Ltd.

616

R. BRZEZIN8KI AND A. PIEKAROWICZ

sequence a n d cleave 25 t o 27 and 25 t o 26 base-pairs 3' t o this sequence, respectively (Bachi et al., 1979; H a d i et al., 1979). Hinflll recogniEes a five-nucleotide sequence and cleaves some 25 base-pairs 3 ' t o this sequence (Piekarowicz et al., 1981). I t has been shown for EcoP15 t h a t b o t h A T P and AdoMet behave as allosteric effectors (Yuan & Reiser, 1978). T h e enzyme forms a complex with unmodified DNA in t h e absence of A T P and Ado^fet. A T P influences this complex a t a late stage of t h e reaction sequence, before D N A cleavage, a n d is affected b y t h e presence of AdoMet (Yuan & Reiser, 1978). The Hinflll enzyme can exist in two different forms; with and without AdoMet bound t o it (Piekarowicz & Brzezinski, 1980). W e shall denote t h e m as HinfITi* and Hinflll, respectively. T h e l a t t e r form can be obtained b y storing t h e enzyme for a considerable period of time a t — 20°C. W e have used t h e purified Hinflll* to s t u d y t h e various stages in the restriction reaction. This enzyme forms complexes with D N A only in the presence of AdoMet. T h e interactions of these complexes with A T P lead t o rapid D N A cleavage, which is a late stage in the reaction sequence. T h e hydrolysis of A T P is probably necessary for D N A cleavage. T h e interaction of A T P a n d AdoMet with t h e Hinflll form of t h e enzyme suggests t h a t it is n o t a n a t u r a l form of this restriction endonuclease.

2. Materials and Methods (a) Nomenclature The names for restriction endonucleases suggested by Smith & Nathans (1973) are further abbreviated as follows. The enzymes from Escherichia coli K and B are referred to as EcdK. and EcoB, respectively. EcoPl and EcoPl5 are the restriction endonucleases coded by bacteriophage PI and plasmid P15, respectively. HinfUI is the restriction endonuclease isolated from Haemophilis influenzae Rf232 (Kauc & Piekarowicz, 1978). (b) DNA preparations Unmodified phage was prepared by thermal induction of the lysogen E. coli ACI867SMSS7. Phage were purified and their DNA extracted essentially as described by Miller (1972). Preparation of ^H-labelled ColBl DNA by amplification in the presence of chloramphenicol (170 ng/ml) from E. coli JC411(ColEl) was performed according to Clewell (1972). The DNA was purified by the cleared lysate technique of Guerry et al. (1973). The plasmid DNA was further purified by dye-buoyant centrifugation in ethidium bromide/CsCl gradients, equilibrated at 20°C for 40 h at 40,000 revs/min in an MSE 50.Ti rotor. (c) Purification of

Hinflll

H. influenzae Rf232 with Hinflll host specificity system has been described (Piekarowicz et al., 1974). These cells were grown in brain/heart infusion medium to A^^Q 0-60. The restriction activity was purified by chromatography on phosphoeellulose and heparin/agarose as described by Kauc & Piekarowicz (1978). The enzyme was stored frozen at — 20°C in 002 M-potassium phosphate (pH 7-4), 0 1 mM-EDTA, 2 mM-2-mercaptoethanol, 20% (v/v) glycerol. Since the freshly isolated enzyme (up to 6 weeks of storage) has AdoMet bound to it and differs from the enzjrme stored for a longer period in respect of the cleavage pattern of ColEl DNA (Piekarowicz & Brzezinski, 1980), we defined Hinflll* as the form of the enzyme with AdoMet bound to it and Hinflll as the free enzyme.

MECHANISM OP ACTION OF THE Hinflll

END0NUCLEA8E

617

(d) Other materials AdoMet, ATP and heparin were obtained from Sigma Chemical Co. The AdoMet was further purified by elution from BioRex 70 with 4 M-acetic acid (Meselson & Yuan, 1968). Adenosine-5'-(j3, y)-imidotriphosphate was purchased from Boehringer, Mannheim. (e) Endonuclease assays The assay mixtures had the following composition: 100 mM-Tris • HCl (pH 8-0), 6 mMMgCl2, 6 mM-2-mereaptoethanol. The reaction volumes, amounts of DNA or ^H-labelled ColEl DNA, amounts of ATP and AdoMet were as indicated in the Figure legends. After different times of incubation at 37°C, reactions were stopped by the addition of 10 ^il of 10% (w/v) sodium dodecyl sulphate. The rate of cleavage with ColEl DNA as a substrate was measured by following the conversion from supercoiled to linear form. The different forms of DNA were then separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Localized DNA bands were excised, solubilized with NCS tissue solubilizer (Nuclear Chicago) and the ' H content of each was determined by liquid scintillation counting. When calculating the extent of cleavage of the circular DNA to the linear form, the fraction of DNA in the linear form (III) was divided by the fraction of DNA in the supercoiled (1), open circular (II) and linear form (III) and denoted as follows: ots/min(III) cts/min(I) + cts/min(II) + cts/min(III) (f) Agarose gel electrophoresis Analytical gel electrophoresis was carried out on 24 cm x 18 cm xO-5 cm slab gels (Sugden et al., 1976) with an agarose concentration of 0-9% (w/v). The gels were run for 6 to 8'h at room temperature at 60 to 80 V. The electrolyte composition was 40 mM-Tris, 36 mMKH2PO4 (pH 7-7) and 1 mM-Na2EDTA. After electrophoresis, all gels were soaked for 10 min in ethidium bromide solution (2 fig/ml) and illuminated with a short-wave ultraviolet light. A red filter was used to photograph the gels. 3. Results (a) The dependence of DNA cleavage on the concentrations ATP and AdoMet

of

I t has been shown t h a t A T P acts as an allosteric effector of EcoPl a n d EcoTlS (Reiser, 1975; Y u a n & Reiser, 1978). These results suggested t h a t A T P m i g h t also behave as an allosteric effector in the / / i n f i l l reaction. The dependence of D N A cleavage on A T P concentration was determined for the two forms of HinflU (Fig. 1). I t is evident t h a t the cleavage reaction is dependent on A T P for b o t h / / m f l l l forms, b u t t h e a p p a r e n t K^ value for Hinilll* is 10~^ M, while t h a t for t h e Hinilll is 5-5 x 10^* M. T h e following criteria support t h e concept of A T P acting as a n allosteric effector for b o t h forms of the enzyme. (1) T h e curves are sigmoidal. (2) T h e eo-operativity index is defined as the a m o u n t of ligand required to yield 1 0 % of A„^^ (Koshland, 1970). T h e value for Hinflll* is 3-4 a n d t h a t for HinflU is —15-0. I n the absence of eo-operativity, this value would be 8 1 . (3) T h e d a t a from Figure 1(a) were replotted according to Hill (1910). The slope of t h e s t r a i g h t line should yield the minimum n u m b e r of A T P binding sites. The value obtained from Figure 1(b) for Hinflll* is 3-73 and t h a t for Hinflll is 1-77, indicating t h a t

318

R. B E Z E Z I l s I S K I A N D A. P I E K A R O W I C Z

-6

-3

-2

I ATP concentration [ M ]

FIG. 1. DNA cleavage by the Hinflll* and HinSLIl enzymes as a function of ATP concentration, (a) The reaction mixtures (30 /xl) containing 10 fd of the HinfTll* or HinOIl enzyme preparation were incubated for 30 min at 37°C using the standard endonuclease assay conditions as described in Materials and Methods. The reactions were carried out with various concentrations of ATP in the presence of 10"* M-AdoMet. (O) Reactions with Hmflll*; ( # ) reactions with Hinflll. The inset shows a plot of cleavage data according to Hill (1910); n = 3-73 for Hinail*; ra = 1-77 for Hinail.

Hinflll* has at least four ATP binding sites, while Hinflll has at least two binding sites. The role of AdoMet in the reaction catalyzed by Hinflll* is more complex, since restriction and methylation occur at the same time. The results presented in Figure 2 clearly show that AdoMet stimulates the rate of cleavage. This suggests that AdoMet might also act as an allosteric effector. The dependence of DNA cleavage by Hinflll* on AdoMet concentration was studied over the range of 10^^ to S x l O ' ^ M (Fig. 3). These values have been normalized by subtracting the amount of DNA cleavage observed with ATP alone. The curve is sigmoidal, as in the case of ATP, and the co-operativity index is estimated at 18-4. The best fit straight line in a Hill plot of the data in Figure 3 has a slope of 1-29. Therefore, Hinflll* fulfils the criteria required for allosteric proteins (Koshland, 1970). I t can be concluded that both cofactors act as allosteric effectors in the endonuclease reaction. The minimum number of AdoMet binding sites in Hinflll could not be estimated precisely because this form of the enzyme cleaves the DNA at more than one site, generating both the linear form of the ColEl DNA and a large number of fragments. Since our method of measuring the extent of cleavage was based on the conversion of superooiled DNA to a linear form, generation of the fragments precluded the use of this method. The additional conclusion emerging from these results is that the cleavage

M E C H A N I S M OP A C T I O N O F T H E Hintin

ENDONUCLBASE

619

100

ElG. 2. Stimulation of DNA cleavage by AdoMet. The reaction mixtures (30 (A) containing 10 lA of the Hinilll* enzyme preparation and 10~ ' M-ATP were incubated at 37°C for the timeB indicated using the standard endonuclease assay conditions as described in Materials and Methods. The reactions were carried out with 10"* M-AdoMet (O) or without AdoMet ( • ) .

reaction is stimulated by the addition of external AdoMet, even though the J^mflll* form has some AdoMet already bomid to it (Piekarowicz & Brzeziiiski, 1980). (b) The effect of heparin on

Hinflll

The experiments with EcoK and EcoFl5 have shown that heparin can be used as a tool in the analysis of the restriction reaction (Yuan et al., 1975; Yuan & Reiser, 1978). Heparin is a polyanionic glycan that competes with DNA for being bound by

-9

I

-7

-6

-5

-4

-3

Log AdoMet concentration [ M ]

P I G . 3. DNA cleavage by the Hinfill* enzyme as a function of AdoMet concentration. The reaction mixtures (30 fd) containing 10 fil of the Hinflll* enzyme preparation and 10"^ M-ATP were incubated for 3 min at Sl'C using the standard endonuclease assay conditions as described in Materials and Methods. The reactions were carried out with various concentrations of AdoMet.

620

R. BRZBZlSSKI AND A, PIEKAROWICZ TABLE 1

Inhibition of H i n / / / / * by heparin

First incubation

Second incubation

Third incubation

Setl (1) DNA + AdoMet + ATP (2) DNA + AdoMet + ATP + ffijiflll* (3) DNA + AdoMet + ATP + ffimflll* + heparin (4) DNA + ATP + ffireflll* (5) DNA + ATP+fl-i«£in* +heparin

% Total DNA cleaved

2-24 9513 3-00 95-00 117

Set 2 (6) DNA + AdoMet + Hinmi* (7) DNA + AdoMet+ fl'mnil* (8) DNA + AdoMet + ffiwflll*

ATP Heparin

Set 3 (9) D N A + i / m f l l l * (10) DNA + flmflll* (11) DNA + ffmflll* (12) D N A + /fmfIII* +heparin (13) DNA + A d o M e t + « a f I I I * + h e p a r i n (14) DNA + ATP+i?s»flII*

ATP Heparin ATP ATP Heparin

ATP

ATP

3-00 95-0 45-0 3-0 95-0 10-61 2-58 3-57 95-00

Set 1: Each reaction mixture of 30 ^1 contained 1 ^g of ColEl DNA and 10 /J1 of enzyme preparation (except reaction 1). The final concentrations of the other components were: ATP, 10"^ M; AdoMet, 10~* M; heparin, 500 fig/ml. Incubation was carried out at 37°C for 10 min and restriction assays were done as described in Materials and Methods. Set 2: These reactions were similar to those described for set 1. The reactions were prerncubated for 30 s, ATP or heparin was added as indicated, and all samples were incubated for 30 s (second incubation) and then for 10 min (third incubation). Set 3: These reactions are similar to those described for sets 1 and 2.

EcoK (Yuan et ah, 1975) and EeoPl5 (Yuan & Reiser, 1978). The results obtained by Yuan & Reiser (1978) showed that EcoPl5 forms a complex with DNA in the absence of AdoMet and ATP, and that these enzyme-DNA complexes are insensitive to heparin inhibition. The effect of heparin on Hinflll appears to be slightly different (Table 1). The first experiments were done with the Hinfill* form of the enzyme. Three sets of experiments were carried out. In set 1, we tested the effect of heparin in the case where it was present in the reaction mixture before addition of the enzyme. The restriction reaction was effectively blocked, regardless of the presence (reaction (3)) or absence (reaction (5)) of AdoMet. In set 2, DNA complexes were formed with HinQll* in the presence of AdoMet, and are denoted as //mfIII* + S (i.e. plus AdoMet). No endonuclease activity was observed with jymfIII* + S in the absence of ATP (reaction (6)). If ATP was added to .ffmflll + S, normal cleavage took place (reaction (7)). Incubation with heparin

M E C H A N I S M O F A C T I O N O F T H E HinUlI

ENDONUCLEASE

621

for 30 seconds followed by addition of ATP (reaction (8)) also resulted in cleavage, although with a slightly lower efficiency. Therefore, Hinflll* can form a complex with DNA in the presence of AdoMet, and heparin does not inhibit the ATPinduced DNA cleavage by such complexes. In set 3, DNA complexes were formed with Hinflll* in the absence of AdoMet, and Hin{III* + S did not undergo endonucleolytic cleavage (reaction (9)). ATP addition (reaction (10)) resulted in a normal cleavage level. Incubation with heparin for 30 seconds, followed by the addition of ATP (reaction (11)), resulted in a very low level of DNA cleavage as compared to the standard reaction (reaction (4)). In a separate set of experiments, it was shown that if heparin was present in the first incubation of Hinilll* with DNA (plus or minus AdoMet), then ATP was added, no DNA cleavage occurred (reactions (12) and (13)). The conclusion that may be drawn from these experiments is that heparin prevents complex formation but, once Hinnil* is bound to the DNA, it becomes heparin insensitive. However, the formation of these fl'mfIII*-DNA complexes insensitive to heparin does require AdoMet. On the other hand, the cleavage reaction can occur in the absence of AdoMet when ATP is present from the beginning of the reaction. In reaction (14), the firat incubation was carried out for 30 seconds, at which time only about 15% of the DNA was cleaved (see Fig. 3). The fact that the addition of heparin does not blocfc further cleavage indicates that some /7iwfIII*-DNA complexes can be formed in the presence of ATP. It is difficult to say whether, under these conditions, formation of a iirmfIII*-DNA complex insensitive to heparin really occurs, because the cleavage of DNA occurs very fast and it is not possible to diptinguiish the formation of such complexes from the cleavage reaction itself. The results presented in Table 2 show that the Hinflll form of the enzyme is unable to form a complex with DNA that is insensitive to heparin, even in the presence of AdoMet (reaction (5)). However, when ATP is present from the beginning

TABLE 2

Inhibition of Hinflll

First incubation (1) (2) (3) (4) (5)

DNA + AdoMet+ATP DNA + AdoMet + A T P + i^«wfIII DNA+AdoMet+ i?mHII +heparin DNA + AdoMet+ATP+HmfIII DNA + AdoMet+ fl'mmi

by heparin

Second incubation

Third incubation

—

— — —

ATP Heparin Heparin

ATP

% Total DNA cleaved 3-72 80-59 3-40 67-58 7-66

Each reaction mixture of 30 |al contained 1 /ig of ColEl DNA and 10 /JI of enzyme preparation (except reaction (1)). The final concentrations of the other components were: ATP, 10~' M; AdoMet, 10~* M; heparin, 500 ftg/ml. Incubation was carried out at 37°C for 30 s, ATP or heparin was added as indicated, and all reactions were incubated for 30 s (second incubation) and then for 10 min (third incubation). Restriction assays were done as described in Materials and Methods.

622

R. B R Z E Z I l S S K I AND A. P I E K A R O W I C Z

of the reaction, some complexes probably can be formed. In reaction (4), the first incubation was carried out for 30 seconds, at which time only about 30% of the DNA is cleaved (data not presented). It can be argued that in this form of the enzyme the release of AdoMet has occurred, and probably also some changes in the structure of the molecules. This interpretation would be consistent with the observation that HinUIl needs a higher concentration of ATP for maximal DNA cleavage as compared to Hinfill*, and that the enzyme shows a weaker cooperativity in the presence of ATP. The fact that .ffmflll* can cleave the DNA in the absence of AdoMet (Kauc & Piekarowicz, 1978) indicates that the first step in the restriction reaction is probably the formation of some initial complex between the enzyme and DNA. Furthermore, we have shown that AdoMet is required by Hinflil* to form a complex with DNA that is insensitive to heparin. Thus, we may assume that AdoMet stimulates DNA cleavage by formation of more stable complexes between the enzyme and DNA, and hence affects the way in which ATP interacts with such a complex to generate DNA cleavage. To prove that, we have compared the effect of ATP concentration on DNA cleavage in the presence or absence of AdoMet (Figs 1 and 4). I t can be seen that in the absence of AdoMet, the apparent K„ value is slightly higher than in the presence of this cofactor, and the enzyme shows a higher level of DNA cleavage in the presence of AdoMet than without this cofactor with the same ATP concentrations. The co-operativity index is estimated at 4-4, and the best fit straight line in a Hill plot of the data in Figure 4 has a slope of 3-01. Therefore, Hinflll* in the ab sence of AdoMet has a minimum of

1000

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40

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Log [ A T P ]

1

1

1

1

-4

-3

-2

-i

1

Log ATP concentration [ M ]

FIG. 4. DNA cleavage by the Hinflll* enzyme as a function of ATP concentration in the absence of AdoMet. The reaction mixtures (30^1) containing 10/j.! of the Hinflll* enzyme preparation were incubated for 30 min at ST^C using the standard endonuelease assay conditions as described in Materials and Methods. The reactions were carried out with various concentrations of ATP in the absence of AdoMet. The inset shows a plot of the cleavage data according to Hill (1910); n = 3-01.

M E C H A N I S M O F A C T I O N O F T H E Hinilll

ENDONUCLEASE

623

three ATP binding sites, which is lower than the number of ATP binding sites calculated for the interaction of the enzyme with ATP in the presence of AdoMet. These results indicate that AdoMet stimulates DNA cleavage by affecting the interaction of the enzyme-DNA complex with ATP. (c) The role of ATP in the endonuclease reaction Thus far we have shown that AdoMet is required by Hintlll* for binding to DNA, and that this cofactor stimulates DNA cleavage by affecting the interaction of the enzyme-DNA complex with ATP. ATP by itself can also stimulate binding of HinfLII* to DNA to form the heparin-insensitive complex. It was also shown that ATP acts as an allosteric effector in the cleavage reaction that triggers DNA cleavage. To find out whether the rate-limiting step in the Hinfl.II* cleavage reaction could be an ATP-induced conformational change in the enzyme bound to DNA, the kinetics of DNA cleavage under standard conditions were compared to that following addition of ATP to a pre-incubation reaction containing DNA, AdoMet and enzyme. A standard reaction is defined as one in which the reaction is initiated by addition of the enzyme to a solution in which all components are present. The results presented in Figure 5 show that the rate of DNA cleavage is faster for the reaction in which ATP was added to the pre-existing DNA-//mfIII* + S complex than for the standard reaction. This result and the fact that Hinfill*+ S cleaves DNA at lower concentrations of ATP clearly suggest that ATP induces a

100

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30

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90

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120

Time (s)

FIG. 5. Kinetics of DNA cleavage by Hinfill*. Two reaction mixes containing 1-0 jig of ColEl DNA in a volume of 250/zl were prepared. The first reaction mixture contained 10~ 3 M-ATP, 10~4M-AdoMet and 2 pi of the HiniUl* enzyme preparation were added to start the reaction, and portions were removed at the appropriate times. They were pipetted into 20 /*! of the NaCl/EDTA solution containing 10% (w/v) sodium dodecyl sulphate (standard reaction). The second reaction mixture, containing DNA, 10~ 4 M-AdoMet and 2 fil of the HinilW* enzyme preparation, was incubated for 2 min at 37°C and then ATP was added to 10~ 3 M to start the reaction. Portions were removed at the appropriate times and pipetted into 20,ul of the NaCl/EDTA solution containing 10% (w/v) sodium dodecyl sulphate (preincubation reaction). Endonuclease assays were done as described in Materials and Methods. ( 0 ) Standard reactions; (O) preincubation reaction.

624

R. B R Z E Z I N S K I AND A. P I E K A R O W I C Z

conformational change in the Hinfill* that allows it to cleave to the 3' side of the recognition sequence. Such an effect of ATP has been shown for the restriction enzymes -EcoK and EcoPlb (Bickle et al., 1978; Yuan & Reiser, 1978). The experiments with EeoK (Bickle et al., 1978) have shown that such conformational change can occur without ATP hydrolysis, although the hydrolysis is needed for endonucleolytic activity. For £'coP15, it was shown that ATP hydrolysis is not needed for ATP-induced conformational change of the ffcoP15 recognition complex of the cleavage of DNA, since they can be elicited by a non-hydrolysable ATP analogue (Yuan et al., 1980). A low level of ATP hydrolysis has been detected that seems to parallel the Hinfill restriction reaction, and that is dependent on the presence of unmodified DNA (Kauc & Piekarowicz, 1978). First, we have focused on the relationship between ATP hydrolysis and DNA cleavage. Adenosine-5'-()3, y)-imidotriphosphate is a non-hydrolysable analogue in which the oxygen between the |3 and y phosphates has been replaced by an imido group (Young et al., 1971). This imido analogue, when substituted for ATP in the restriction reaction with ColEl DNA, will not sustain DNA cleavage (Table 3, reaction (1)) even after the formation of the DNA-7/mfIII*-AdoMet complex (reaction (3)). This is in sharp contrast to Ec6P\5, where this imido analogue sustains DNA cleavage (Yuan et al., 1980). Previous experiments have shown that Hinflll turns over in the endonuclease reaction. Our results could suggest that ATP hydrolysis is required either for DNA cleavage or to act as an allosteric effector able to induce conformational changes in the enzyme bound to the DNA. It could also be required in order for Hinflll* to act catalytically by releasing the enzyme from the DNA. Such a dissociation of the enzyme from the recognition complexes has been observed for EcoPlB (Yuan et al., 1980). However, the fact that in the presence of the imido analogue of ATP even a limited cleavage reaction was not observed, argues against requirement of ATP TABLE 3

The effect of adenosine 5'-(P, y)-imidotriphosplmte on the restriction activity of

mnflll

First incubation (1) (2) (3) (4)

DNA + AdoMet + imido-ATP + DNA + AdoMet + ffireflll* DNA + AdoMet + /?TOflII* DNA + AdoMet + ffinflll*

(5) DN4+AdoMet + / / m n i I *

Second incubation

Third incubation

Heparin Heparin Imido-ATP

ATP Imido-ATP Heparin, imido-ATP ATP

ffmnil*

Imido-ATP

% Total DNA cleaved 3-20 45-0 30 2-85 92-0

Each reaction mixture of 30/nl contained 1 /xg of ColEl DNA and lOfil of enzyme preparation. The final concentrations of the other components were: ATP, 1 0 " ' M; imido-ATP, 1 0 " ' M; AdoMet, 1 0 ~ * M ; heparin, 500 jug/ml. Incubation was carried out at 37°C for 30 s, ATP, imido-ATP or heparin were added as indicated, and all reactions were incubated for 30 s (second incubation) and then for 10 min (third incubation). Restriction assays were done as described in Materials and Methods.

MECHANISM OF ACTION OF THE Hinail

ENDONUCLEASE

625

hydrolysis only in order for Hinflll* to act catalytically. Moreover, the fact that the presence of the imido analogue of ATP makes the DNA-//mfIII*-AdoMet complex susceptible to heparin (reaction (4)), while such a complex in the presence of ATP (Table 2, reaction (4)) is still insensitive to heparin, could suggest that the analogue releases the enzyme from the DNA without triggering the complex to cleave the DNA. To obtain more information about the role of ATP in the particular steps of the reaction, the rate of DNA cleavage was measured following addition of ATP to a reaction mixture pre-incubated with DNA, AdoMet, imidoATP and Hinnil*. It was shown (data not presented) that the rate of cleavage following the addition of ATP to the reaction mixture containing the imido-ATP is comparable to that without the analogue (Fig. 5), which would suggest that the hydrolysis of ATP is required to induce a conformational change in the enzyme, allowing it to cleave the DNA. However, it is still an open question as to whether the hydrolysis of ATP is required for the cleavage of DNA. 4. Discussion The early steps in the Hinflll restriction reaction have been studied in vitro. Both ATP and *S-adenosyl-L-methionine have been shown to act as allosteric effectors in the endonuclease reaction. Hinflll* has a minimum of four binding sites for ATP in the reactions carried out in the presence of AdoMet. It has a minimum of two binding sites for AdoMet. However, since Hinflll* has some AdoMet bound to it (Piekarowicz & Brzezinski, 1980), it is possible that the enzyme has more AdoMet binding sites than this. In order to study the interaction between the enzyme and DNA, we used the glycan heparin, which inhibits the interaction between the enzyme and DNA but, once //iwfIII*-DNA complexes have been formed, they become insensitive to heparin inhibition. This permitted us to study the characteristics of these complexes. As opposed to the other type III restriction enzymes (Yuan & Reiser, 1978), Hinflll* requires external AdoMet to form, with unmodified DNA, the complexes resistant to inhibition by heparin. Since Hinflll*, as well as Hinflll, can cleave the DNA in the absence of AdoMet, the first step in the cleavage reaction would be the formation of the initial complex between the enzyme and DNA. This complex would be capable of cleaving the DNA after the addition of ATP. However, the presence of external AdoMet would result in the formation of the complexes ( + S), which are insensitive to inhibition by heparin, able to cleave the DNA at lower concentrations of ATP and having faster kinetics of DNA hydrolysis following the ATP addition. The rate-limiting step in the cleavage reaction would be an ATP-induced conformational change in the enzyme bound to the DNA. In this respect, Hinflll* behaves similarly to -E'coPl and EcoPl5, which also form complexes (-l-S) having faster kinetics of DNA hydrolysis, and which cleave the DNA at lower concentrations of ATP than do — S complexes (Yuan & Reiser, 1978). Hinflll* has some AdoMet bound to it (Piekarowicz & Brzezinski, 1980) and thus can form complexes that could be defined as + S complexes. However, this amount of AdoMet is either too low for the formation of the true -|-S complexes or the AdoMet molecules bound to Hinflll* are used only for the methylation and cannot introduce the conformational changes in the

626

R. BRZEZINSKI AND A. PIEKAROWICZ

enzymes molecules t h a t would enable them to form heparin-insensitive complexes with D N A . I t seems t h a t Hinflll is an u n n a t u r a l form of the enzyme. I t needs very high concentrations of A T P for DNA cleavage, a n d it is unable t o form a more stable complex with DNA, even in the presence of external AdoMet. I t is possible t h a t the storage of the enzyme results in the release of internal AdoMet and in some conformational changes, resulting in the molecule n o t being affected by t h e addition of external AdoMet. The use of an A T P analogue enabled us to study the interaction of A T P with recognition complexes, and to show some differences between HinHIl* and other t y p e I I I restriction enzymes. F o r EcoPl5, A T P hydrolysis does not appear to be required for D N A cleavage, since a limited reaction was observed when A T P was replaced b y its imido analogue (Yuan et al., 1980). I n t h e case of / / i n f i l l * , no restriction reaction was observed when the imido analogue was used in the place of A T P . This would suggest t h a t for Hinflll* there is a more stringent requirement for A T P hydrolysis during the restriction reaction t h a n for EcoPl or EcoPl5. T h e low level of A T P hydrolysis t h a t has been detected with HinUll* seems t o be associated with the conformational change of the enzyme t h a t allows it to cleave a t sites to t h e 3' side of the recognition sequences. I t m a y be t h a t A T P is required for some translocation of the DNA during the cleavage reaction or for the interaction of the enzyme in the recognition complex with the cleavage site. This would explain some observations showing t h a t linear DNA molecules are cleaved less efficiently t h a n supercoiled ones, and t h a t certain sites are more susceptible for endonuclease action or t h a t DNAs carrying only one or two recognition sites are n o t cleaved although t h e y can be m e t h y l a t e d (A. Piekarowicz, unpublished observations). I n conclusion, the HinUll enzyme, together with EcoPl and EcoPl5, represent the t y p e I I I restriction enzymes, although some details of t h e mechanisms of t h e cleavage reactions are different for HinUll. This work was supported by the Polish Academy of Sciences (research project 09.7.2). Some experiments were done during the tenure (by A.P.) of a European Molecular Biology Organization short-term fellowship. We are grateful to Dr T. A. Bickle for helpful discussion and for critical reading of the manuscript.

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