[17] Fluorometric assay for specific (nickases) and nonspecific endodeoxyribonucleases

[17]

FLUOROMETRY OF ENDONUCLEASES

255

32p from the 5'-terminus is released more rapidly than unlabeled total nucleotide. The preferential release of label from the 5'-end is slower than from the 3'-terminus. The UV-exonuclease is therefore capable of degrading denatured DNA from either the 3'- or the 5'-terminus, in an exonucleolytic manner, to yield 5'-mononueleotides as its final degradation products.

[ 17] F l u o r o m e t r i c A s s a y f o r S p e c i f i c ( N i c k a s e s ) a n d Nonspecific Endodeoxyribonucleases

By C. PAOLETTIand JEAN BF_~NARDLE PECQ Principle Ethidium bromide (EB) has been shown to be a double-stranded DNA intercalating dye bringing about a rotation of the base pairs along the DNA duplex axis, and, consequently, an unwinding of each strand. When EB saturates an open DNA, 1 the two strands are able to rotate around each other since there is a topological freedom at both ends of the molecule (linear DNA) or at the breaks along it (circular DNA). A maximum unwinding is reached for a saturating amount of EB. rsatop, is defined as the amount (number of molecules) of saturating EB bound to open DNA per nucleotide. When EB saturates closed circles, the same events occur, but the absence of any free rotating end or any swiveling point results in a topological constraint imposed upon the molecules such that the unwinding cannot proceed to the maximum extent for open DNA. Consequently, r s a t c, the amount (number of molecules) of saturating EB bound to closed circles per nucleotide, is smaller than rsatop. One unique break on a closed circle provides a swivel point that relieves the molecule from the topological constraint and allows it to rotate and permits the binding of more EB. In other words, the first break on a closed circle results in an increase of the amount of dye that can be bound at saturation. Such a correlation yields a convenient way to evaluate, on a homogeneous population of closed circles, the number of circular molecules that have been opened and therefore the number of nicks induced by an endonuclease or any breaking agent. 1Covalently closed double-stranded circular DNA will be referred to as "closed circles"; circles with at least one break, as well as linear molecules, are "open DNA."

256

REPAIR, RE~RICTION, AND INTEGRATION OF DNA

[17]

The fluorescent properties of EB 2 has been used as a convenient tool to follow its binding to DNA. The increase of fluorescence intensity I - I~B when free EB is bound to DNA is proportional to r, the amount of dye bound by nucleotide on the DNA. I--IEB

= ~XQXEXrXS

(1)

I~.B and I being the fluorescence intensities of the EB solution without polymer and after addition of the polymer; a, a proportionality coefficient constant under defined experimental conditions; Q, the EB quantum efficiency of fluorescence in the EB-DNA complex; ~, the absorption coefficient of the bound dye at the exciting wavelength; and S, the DNA concentration. A theoretical study 8 has shown that the changes of fluorescence intensity of DNA circles exposed to a DNase and then saturated with EB can be correlated to the parameters of the system depending on the mode of action of the nuclease. For a nonspecific nuclease, i.e., an enzyme that can hydrolyze a great number of bonds along a DNA molecule, the substrate concentration (breakable b o n d s - broken bonds) does not change during the time necessary to open all the circles because the number of broken bonds (a few per molecule) is negligible compared to that of the breakable bonds. lop -- It

v X t X M.

n Io--~ i o =

SXN

(2)

Io, It, and Io, being the fluorescence intensities at time 0 when the enzyme

is added on the DNA, at time t and at time oo when all the circles are open DNA; v, the rate of action of the DNase (number of breaks performed by the enzyme per milliliter per minute); t, time elapsed since the beginning of the enzyme action on DNA; M , , DNA molecular weight; S, DNA concentration (g/ml) ; N, Avogadro's number. For nuclease with a large Km (such as pancreatic DNase), Eq. (2) can be written: Vm~ X t X M,, K,,XN

(3)

In lop -- It __. Vmax X t X Mw l o p - - Io S × N

(4)

I o p - - It In Iop Io

=

and for a nuclease with a small K,~

2j. B. Le Pecq and C. Paoletti, J. Mol. Biol. 27, 87 (1967). C. Paolettl, J. B. Le Pecq, and I. R. Lehman, J. Mol. Biol. 55, 75 (1971).

[17]

FLUOROMETRY OF ENDONUCLEASES

257

The slope of the fluorescence changes, as expressed in Eqs. (3) and (4), are linearly proportional to V~x, i.e., to enzyme concentration, to time, and to DNA molecular weight; it can or cannot be correlated to the substrate concentration, depending on the Km of the enzyme. For a "nickase," i.e., a specific endonuclease attacking DNA at one or very few specific sites along each molecule, eventually on one strand only, one gets: In 1o, - I~ = Vm,x × t I o , -- Io

(5)

K,.

In this case, the log slope of fluorescence changes would not depend on the DNA molecular weight. Procedure

Choice and Preparation of D N A Substrate

The choice of the substrate depends on the ease and the yield of its preparation, the sensitivity and accuracy desired, and its specificity toward the studied nuclease (for example, unmodified X DNA for the restriction enzyme). The higher the molecular weight of DNA, the higher the sensitivity [see Eqs. (3) and (4) ] ; the greater the difference between Iop and Io, the better the accuracy. For a given species of D N A , / o , - - I o is proportional to rsatop - - r s a t c ; since it is known that rsatop is constant for any open DNA, the smaller is r s a t c, the greater is Iop -- It. The table gives rsatc for different kinds of DNA. PM2 bacteriophage of Pseudomonas has been the most convenient source of closed circles in our hands. The procedure for growing the phage and extracting DNA is described by Espejo et. al. 4,5 One procedure for the preparation of closed circles is as follows: 0.5 ml of PM2 phage suspension (OD~6o about 50) is diluted in 5.4 ml o f SCC (0.15 M NaC1, 15 mM sodium citrate), 0.1 ml of 0.1 M EDTA (pH 7.5), and 1.5 ml of a 5% Sarkosyl NL 97 (Geigy) solution in 10 mM EDTA pH 7.5. After 10 minutes at room temperature, 2.8 ml of a 4 mg/ml EB solution in SCC, 1 mM EDTA, and 9.9 g of CsC1 are added (refractive index---- 1.3890). Centrifugation is performed at 35.000 rpm for 70 hours at 4 ° in an SW 39 rotor. The lower bands are collected; EB is eliminated by two washings with butanol (2:1 v / v ) ; finally, a dialysis is performed against the *R. T. Espejo and E. S. Canelo, Virology 34, 738 (1968). R. T. Espejo, E. S. Canelo, and R. L. Sinsheimer, Proc. Nat. Acad. Sci. U.S. 63, 1164 (1969).

258

[17J

REPAIR, RESTRICTION, AND INTEGRATION OF DNA

FLUORESCENCE OF ~'~B BOUND AT SATURATION ON DIFFERENT CLOSED CIRCULAR D N A

Reading on the Zeiss transmittance scaleb Nature of DNA A. Open DNA~ B. Closed circles DNA ¢X174 RFI ¢X174 RFI k k Trypanosome PM2 SV40~

Origin of DNA

r.~to~

(ire -- IEB)

Any

0.20

100

Infected bacteria Synthetized in vitro ~ Superinfected bacteria Hershey circles ~ Escherichia coli joining enzyme Kinetoplasts Virions Virions

0.15 0.10 0.15 O. 12

75 50 75 60

0.12 0.15 0.13

62 73 65

a Number of molecules of EB bound at saturation to DNA per nucleotide. b IEB, Iop, and Ic are the fluorescence intensities of the EB solution without DNA, with 100% open DNA and with 100% closed circles. The readings given in this column have been standardized for I o p - I~s = 100 division of the fluorometer scale. c j. B. Le Pecq and C. Paoletti, J. Mol. Biol. 27, 87 (1967). d From W. Bauer and J. Vinograd, J. Mol. Biol. 33, 141 (1968). M. Goulian, A. Kornberg and R. L. Sinsheimer, Proc. Nat. Acad. Sci. 58, 2321, 1967. buffer used in the nuclease assay. All the materials must be sterile. The yield of closed D N A is around 5 0 ~ . Besides the usual ultracentrifugation methods, the easiest way to know the proportion, p of closed D N A circles in a mixed population of closed and nicked molecules is the following: add 5 ~l of pancreatic D N a s e at 10.2 ~gfml to 100 ~l of the D N A at about 5 ~g/ml in the optimal buffer for this enzyme (cacodylate 50 m M ; MgC12, 4 m M ; CaC12, 2 m M ; p H 6.3). After 15-20 minutes at 37 ° add 700 ~l of EB-SS and measure the fluorescence intensity Iop, as described below; Ip and IEB are the control fluorescence intensities of aliquots devoid of enzyme and of DNA. Io9 - I , 0.20 P = I o p - IEB X 0.20 -- rsat c Values for rsat c can be taken from the table. Fluorometry F l u o r o m e t e r . The procedure will be described for the fluorescence attachment ZFM4 of the Zeiss spectrophotometer P M Q II, but several other fluorometers could be used with, at least, the same efficiency.

[17]

FLUOROMETRY OF ENDONUCLEASES

259

A 1-cm quartz microcuvette can be used; at least 700 ~l of solution are necessary. The reproducibility of the method being very critical, it is advisable to use only one cuvette tightly stuck into the holder for a series of measurements. Monochromatic excitation light (Xexc---- 365 m~) is provided by a mercury lamp through a filter (M 365). Standard monochromator and photomultiplier are used to measure the intensity of the fluorescent light emitted through the bottom of the microcuvette. Any reflection on the walls of the microcuvette must be avoided; therefore, a 2-mm wide slit is put after the M 365 filter; the width of the slit before the monochromafor is adjusted to about 0.5 mm. The emission is recorded at 590 m~. The readings are performed on the transmittance scale at the maximum amplification ()< 10). Standard EB Solution (EB-SS). The standard EB solution that is saturating for DNA concentrations used in this assay is made up of EB (1 ~g/ml) dissolved in 10 mM (pH 7.8), Tris.HC1; 20 mM NaC1, and 5 mM Nag EDTA. A buffer is used although the fluorescence intensity does not change very much with pH. 2 NaC1 concentration is chosen so that only the first EB site of DNA is able to bind the dye and EDTA stops the enzyme reaction. Moreover, the dilution of the aliquot from the reaction mixture into EB-SS, the decrease of temperature (EB-SS is kept at 0°), as well as the inhibitory properties of EB toward most nucleic acid enzymes lead to the same result. Control o/ Temperature. The readings can be performed at any constant temperature between 10 and 25 °. The excitation spectrum of EB undergoes slight modifications with changes of temperature, and the intrinsic binding constant, k, as well as the other thermodynamic constants, AS, AH, AG, for binding of EB to DNA, depend on temperature. Moreover, r.~atop- rsatc, on the value of which this method depends, is proportional to the duplex winding number of the DNA, which is itself a function of temperature. Consequently, the control of temperature during the fluorometric readings is a critical point; for a given solution, the lower the temperature, the higher the intensity of the fluorescence. Sensitivity. For EB-SS in the usual buffers, the readings lie around 12 under our previous conditions. A deflection of 1-2 divisions on the scale is induced by a DNA concentration of 8 X 10.3 t~gJml depending on the mercury lamp. Usually, the concentration of closed DNA into EB mixture is adiusted as to set a deflection of about 80 divisions after substraction of the background due to EB-SS alone; 0.26-0.56 ~g of DNA or 0.8-1.6 nmoles of DNA nucleotides must be mixed with the 700 ~1 of EB-SS to get a convenient deflection. This means that the DNA reaction mixture

260

REPAIR, RESTRICTION, AND INTEGRATION OF DNA

[17]

can be between 1 and 500/~g/ml, 200 #1 to 1 ~1 of it being added to 700 /~l of EB-SS to read in the fluorometer. For further details on the sensitivity of this method, see Le Pecq and Paoletti. 6 It should be noted that the intensity of the light emitted by the mercury lamp is not constant and varies from one bulb to another. Stability and Reproducibility. The Hg bulb must be checked against a usual fluorescent glass standard source. For unknown reasons, fluctuations in the excitation light intensity sometimes occur during measurements. In most cases, after a few minutes, the bulb intensity comes back exactly to the same level as before fluctuation; otherwise, a complete set of measurements has to be run again. Rigorous cleanliness of glassware and cuvettes is an absolute requisite for this method; many chemicals are potent fluorescent agents or fluorescence modifiers, most often quenchers, even as traces. For instance, a detergent commonly used in our laboratory has been found to emit a blue fluorescent light at low concentration. In practice, any glassware must be acid-washed. Even at the highest amplification level of the Zeiss speetrofluorometer, its stability is such, if properly selected (i.e., plot III) as to allow a confident reading with a precision better than 0.2 division on the scale. EB is a light-sensitive dye; EB solutions must be kept in the dark and at 0% The controls must be carefully run, since many substances used in enzyme studies (glycerol, nucleotides, BSA, etc.), as well as changes in ionic environment, modify fluorescence?

Assay At time 0, the dilution of endonuclease to be tested is added to the DNA solution (volume between 500 and 1500 ill) ; at different times after this addition, a given volume of the assay mixture (most often 100 ~l) is pipetted out; this volume is mixed up to 700/,1 or 800 #l of EB-SS at 0 °. Depending on the precision expected for the kinetics of opening the circles, 5-15 aliquots are removed over a time calculated for the reaction to come to nearly total completion (at least one average nick on each circle) ; this time must be between 20 and 150 minutes, most often around 50 minutes. After the end of the experiment, the set of samples is moved from 0 ° to a water bath whose temperature is that of the fluorometer cuvette, and the fluorescence readings are performed as previously described.

Expression of the Results Figures 1 and 2 exemplify the data obtained for the opening of closed circles of ADNA and PM2 by pancreatic DNase. ej. B. Le Pecq and C. Paoletti, Anal. Biochem. 17, 100 (1966).

[17]

261

FLUOROMETRY OF ENDONUCLEASES Time in minutes (for X DNA) 25

50

T5

I00

20

15

I

I0

/

o/ P

20 z

I /

/ # 140 I

0

lO

20

3~0

40

Time in minutes (for PM2 DNA)

Fin. 1. Kinetics of opening D N A closed circles by pancreatic DNase. Linear representation. (a) Bacteriophage XDNA (O, (D, - - -). To 1275 #1 of substrate solution (XDNA : 4 ~g/ml; 41.6% of closed circles in 10 m M Tris, p H 7.8, 2 mM M g C l : ~ - 1 m M E D T A ) was added, at time 0, 1 #1 of a pancreatic/)Nase solution (2 X 10-4 m g / m l in 10 m M potassium phosphate buffer pH 72; 5 m M MgCh; 0.1 m g / m l BSA). Two different experiments (dotted circles and open circles) were carried out. Temperature, 37°C. Samples (200 #l) of reaction mixture were taken out at 6 different times in 700 ~l of EB-SS. Fluorometric measurements were performed as described in the text. Io, It, and Iop are the fluorescence intensity increase after addition of EB-SS to 200 #l aliquots, respectively, at time 0, at time t, and at time oo (when the plateau is reached). Io was 87.5; I o p - - I o was 162; h ~ - ~ 12,8 minutes. IEB (fluorescence background) of ED-SS added to D N A buffer and enzyme solvent was 8.7. The reaction mixture contained 2 X 10-' #g of enzyme (concentration: 1.6 X 10-4 ~g/ml). (b) Bacteriophage PM2 D N A ( + ) , To 1030 #1 of substrate solution (PM2 D N A : 72 ~g/ml; 100% of closed circles) was added, at time 0, 10 #1 of a pancreatic DNase solution (2,5 X 10-4 mg/ml). Temperature, 30°C. Samples (90 #l) of reaction mixture were taken out at different times in 800 ~l EB-SS. Fluorometric measurements were performed as described in the text. Buffers for D N A and DNase are identical to those used with XDNA (see above). Io was 70/,; lo,--Io was 21.6; I~B was 122; tl~ ~ 2.6 minutes. The reaction mixture contained 2.5 >( 10-' ~g of enzyme (concentration: 2 . 4 X 10-' ~g/ml).

262

REPAIR, RESTRICTION, AND INTEGRATION OF DNA i

[17]

i

I00,

50

I

2O

Closed circles in % of inificfl closed circles

10 0

I0

20

30

40

Time in minutes (XDNA)

Iop-If Iop-Io x I00 I00

f

'

'

;

50 . . . .

,, 2O

t~ = 2.6 rain ,o

'i

,

5

\

i'o

Time in minul"es (PM2 DNA)

Fie. 2. Kinetics of opening DNA closed circles by pancreatic DNase : semilogarithmic representation. Curves of Fig. 1 have been drawn on a linear log scale.

These data can be plotted in linear coordinates, yielding a exponential curve (Fig. 1), or in semilog coordinates, yielding a linear representation

(Fig. 2). tl/2 is defined as the time when half the maximum increases of fluorescence has been obtained; i.e., the time where 5 0 ~ of the initial number of closed circles have been opened, tl/2 is inversely proportional to the slope of the semilog curve and to the amount of enzyme. One fluorometric unit (FU) of endodeoxyribonuclease activity is defined as the amount of enzyme which induces on D N A 1.62 X 101° breaks per minute per milliliter in a D N A solution at 5 #g/ml in the experimental conditions described on Fig. 1. Such a definition was chosen for experimental convenience; t~/2 for 1 F U is 4 minutes when X D N A (Mw - - 32 X 10e) is used as a substrate. In other words, 1 F U induces 0.17 average break per minute per X D N A molecule or 0.69 average break over t~/2. If the concentration of the D N A solution used as a substrate is modified by a factor x, tl/2 for 1 F U will

[17]

FLUOROMETRY OF ENDONUCLEASES

263

be x X 4, provided that v does not depend on (S), i.e., that K,, of the enzyme is lower than one-tenth of S. However, it should be noted that for the same amount of enzyme, tl/2 will be constant, whatever the DNA concentration, if the DNase displays a relatively high Kin, i.e., if v is linearly proportional to S; this applies to pancreatic DNase (Kin ~ 1 mgJml). tl/2 is inversely proportional to the molecular weight of DNA; in other words, T1/2 for 1 FU would be 4 X (32 X 10G)/Mw when a DNA displaying a molecular weight of M , is used as a substrate. Use of the Method

Determination o] Endonuclease Concentrations From Eqs. (2) and (5) it is apparent that, for both types of endonueleases--nonspeeific ones or nickases the fluorescence changes versus time yield a straight line whose slope should be linearly proportional to the velocity of the enzyme action, i.e., to the concentration of the enzyme. Such a relationship has been verified for the pancreatic DNase (Fig. 3). Consequently, this method leads to the determination of any unknown concentration of endonuclease from the kinetics of opening closed circles when a proper calibration curve has been established with a given DNA.

Determination o] the Molecular Weight o] Closed Circular DNA From Eq. (2), it appears that, for any nonspecific endonuclease, the amount of enzyme necessary to open the DNA circles at a given rate should be inversely proportional to the molecular weight of the DNA; the greater the molecular weight, the less enzyme is needed to open DNA. Consequently, from a calibration curve established with an enzyme at a given concentration in well defined conditions or even from only one standard circular DNA of known molecular weight, any unknown molecular weight can be determined from one kinetic experiment only. A calibration curve is given for the pancreatic DNase in 10 mM TrisHC1, pH 7.8; 2 mM MgC12; 1 mM EDTA; BSA, 1 mgflml (Fig. 4).

Determination of the Turnover Number for Endonucleases This method is the only one which is able to express Vmax of endonucleases in true hydrolytic events (breakage of the phosphodiester bonds per unit of time). Therefore, if the molecular weight of the enzyme is known, the turnover number can be easily calculated.

264

REPAIR, RESTRICTION, AND INTEGRATION OF DNA I

I

i

[17]

I

1.5

3.5

u

"~

65 ~

0 I,I-

0.5 19.0

0

33.0 73.0

;

' 15 Pancreatic DNase

2'5

(y.glmlxlO -4)

FIG. 3. Relationship between the rate of fluorescence changes and the concentration of pancreatic DNase. To five reaction mixtures, each being 400 gl of substrate solution (k D N A 6.8 #g/ml; closed circles ~-~ 37.8%) were added at time 0, 1 #1 and 3 gl of pancreatic DNase solution at 4 X 10-~ #g/ml and I, 3, and 5 gl of the same pancreatic DNase in solution at 2 X 10-1 #g/ml. Temperature-37°C. Aliquots of 75 #1 were taken out at appropriate times between 2 and 150 minutes, depending on the enzyme concentration, and were mixed with 700 #1 of EB.SS. The measurements were carried out as described in the text; tl/2 was measured on each of the five semilogarithmic representations of the data as described for Fig. 1. Io was 103.3; I o p - Io was 112; I t s was 16.0. For buffers of D N A and DNase, see Fig. 1.

Iu]ormation on the Mechanism o] Action and the Purity o] an Endouuclease In principle, this method is strictly specific for the endonucleases, and no pure exonuclease would be recorded in this assay; but, as soon as a closed circle is opened by an endonucleolytic hydrolysis, it becomes sensitive to some exonucleases; when a nonlinear semilog plot (see Fig. 2) is obtained, one must suspect the existence of a exonueleolytic component in the enzymatic activity which releases some mono- or oligonucleotides from the DNA secondary structure at the points of endonucleolytic breakage. Such a removal results in a decrease of the EB binding ability to DNA, since this binding is specific for double-stranded structure. Consequently, there occurs a decrease of the fluorescence in-

[17]

FLUOROMETRY OF ENDONUCLEASE$

265

Tryponosomo • kinetoplost "~'/~

o_×I00

-ft.

T

3ovine / '~ -poplnomo DNA / (4.9x I~//

i~..,,=-PM2DNA(6.0XlO6) ~" .~ XDNA(32x106) 20 50 80 PancreaticDNaseconcentration(tzg/mlX104) FIG. 4. Relationship between the rate of opening of closed circular D N A and its molecular weight. T h e three first experiments (X, papilloma, and trypanosome D N A ) were performed with the same pancreatic D N a s e solution (0.2 #g/ml). The last one ( P M 2 D N A ) was performed with another batch. Aliquots were mixed at different times with 800 #1 EB.SS. F o r buffers of D N A and DNase, see Fig. 1. T e m p e r a t u r e was 30°C. (a) On circular k D N A (Mw ~ 32 X 10'). To 1850 #l of circular k D N A (5.0 ~g/ ml; 41.6% closed circles) was added, at time 0, 3 #1 of enzyme solution. Aliquots: 150 #1. Ion---119.4 (this value was read at a lower amplification); Iop--Io----15.0; IE~ -~ 22.9. Pancreatic DNase concentration : 3.2 X 10-~ #g/ml ; h/2 : 5.5 minutes. (b) On bovine papilloma virus D N A (Mw ~ 4.9 X 10~). To 750 #1 of bovine papilloma virus D N A (4.3 # g / m l ; 90-100% closed circles) was added, at time 0, ? #l of enzyme solution. Aliquots: 100 #1. Io : 90.9; Iop - - Io : 14.0; I~B --~ 12.5. Pancreatic D N a s e concentration : 2.14 X 10-3 # g / m l ; tl~ ~ 2.0 minutes. (c) On trypanosoma kinetoplast D N A (Mw -~ 0.9 X 10~). To 520 #l of kinetoplast D N A (3.6 ~ g / m l ; 90-100% closed circles) was added, at time 0, 36 ~1 of enzyme solution. Aliquots: 10(} #l. Io ~ 82,$ ; lop - - Io ~ 20~ ; I~B -~--14.2. Pancreatic DNase concentration : 1.13 X 10-2 ~ g / m l ; h/~ ~-~ 4.0 minutes. (d) On P M 2 bacteriophage D N A ( M , z 6.0 X 10~). To 1030 ~l of P M 2 D N A (35 # g / m l ; 100% closed circles) was added, at time 0, 10 ~1 of a DNase solution (025 #g/ml). Aliquots: 90 #1. Io ~ 70.5; I o p - - Io--~ 21.6; I~B ~ 12.2. Pancreatic DNase concentration: 2.4 X 10-3 # g / m l ; h/~ ---- 3.0 minutes.

tensity which parallels the increase of fluorescence intensity due to the opening of circles. The existence of a very important exonucleolytic component in the activity of the endonuclease of E. coli has been established through the use of this fluorometric assay2

266

REPAIR) RESTRICTION, AND INTEGRATION OF DNA

[17]

In]ormation on the Specificity o] an Endonuclease

All the known joining enzymes so far described have the same absolute requirement for single-strand breaks possessing adjacent 3'-hydroxyl and 5'-phosphate. One can expect such enzymes to greatly reduce the apparent rate of opening of the closed circles by an endonuclease which yields this type of break on one DNA strand, such as pancreatic DNase, if the two enzymes work together. In contrast, no change in this rate will appear if the specificity of the endonuclease does not match that of the joining enzyme. These expectations have been fulfilled for pancreatic DNase and E. coli endonuclease. Therefore, this method provides an easy and quick tool to get preliminary information on the specificity of an endonuclease; all the technical details have been described elsewhere2 Search ]or a New Nickase or Endonucleolytic Activity

A procedure will be given, as an example, for the search for E. coli endonuclease II type activity, which is normally present at a very low concentration in uninfected cells ~ and is induced by T4 bacteriophage2 A sonicate of wild T4 late infected cells in 50 mM glycylyglycine, pH 7.0, 10 mM fl-mercaptoethanol, and 1 mM EDTA (protein concentration about 2 mg/ml) is diluted out in the same buffer (1:50 v/v). Two volumes of this dilution are added to 1 volume of boiled RNase (100 ~g/ml) and incubated at 37 ° for 30 minutes. When 5 ~l of the RNase-treated solution is added to 1000 ~l of circular closed DNA (4 ~g/ml; 39% of closed circles in 10 mM Tris.HC1, pH 7.8; 2 mM MgC12; 1 mM EDTA), tlz2 is found equal to 11 minutes, the protein concentration in the assay mixture is around 0.1 ~g/ml; the same activity would have been obtained (Fig. 3) for a pancreatic DNase concentration of around 7 X 10"4 ~g/ml. Main Features of the Fluorometric Assay Advantages o] the Assay Sensitivity. This fluorometric assay, when bacteriophage X closed circles are used as a substrate, can pick up one endonucleolytic break out of 105 phosphodiester bonds. In other words, this assay can detect and

E. C. Friedberg and D. A. Goldthwait, Cold Spring Harbor Syrup. Quant. Biol. 33, 271 (1968). s j. Hurwitz, A. Berku, M. L. Gefter, and M. Gold. J. Cell. Physiol. 70, Suppl. 1, 181 (1967).

[17]

FLUOROMETRY OF ENDONUCLE~SES

267

measure the concentration of an amount of pancreatic DNase which is about 6000 times smaller than the amount detected by any method relying on the collapse of the DNA secondary structure. In absolute terms, a concentration as low as 10.5 ~gJml can be detected if the environmental conditions are chosen so as to provide the best conditions for the enzyme action. With PM2 DNA, which is the most convenient substrate, the sensitivity is still 1000 times higher than that provided by the conventional methods. The sensitivity ratio between the conventional and the fluorometric methods depends on the mode of action of the enzyme and does not apply to all nucleases. It should be identical for pancreatic DNase and for any nuclease that is a true endonuclease, i.e., that performs only random breaks on the polymer. On the contrary, it should be reduced f o r an endo-exonucleolytic type of attack, such as by E. coli endonuclease P ; in this case, the removal of a large piece of nucleotide, which occurs after any endonucleolytic break, amplifies the mere collapse of the structure induced by the endonucleolytic break and increases the sensitivity of the conventional methods; the fluorometric method applied to E. coli endonuclease I is about 150 times more sensitive than any other method based on the collapse of the secondary structure. When compared to more sensitive physical methods, among which viscosimetry is probably the best, 9 the fluorometric technique is still 400 times more sensitive for true endonucleases. Finally, the biological methods display a sensitivity close to that of the present one, but probably less. One break on a molecule is not able to destroy the total transforming ability of the DNA. This property depends only on the integrity of the part of the molecule which carries the gene controlling the function which is looked for in the transforming test; such a break must be located in this region of the molecule to be efficient. Nevertheless, any comparison from this standpoint alone can be misleading, since it has been shown that a few breaks only preclude efficient integration of the transforming material into the recipient genome.

Capability ]or Recording Initial Hydrolytic Events The measurement of the opening rate of the closed circles is the only convenient method which expresses the initial nucleolytic events induced by an endonuclease; protonometry is also able, in principle, to achieve this aim but cannot be routinely used at such a sensitivity. All the other usual methods for measuring the DNase activites are gJ. B. Le Pecq and D. Bourgoin, Biochim. Biophys. Acta 80, 173 (1964).

268

REPAIR, RESTRICTION, AND INTEGRATION OF DNA

[17]

empirical, and their results cannot be expressed in absolute terms. Moreover, they express phenomena, usually the collapse of the secondary structure and the transformation of polynucleotides in acid-soluble products, which are the remote consequence of the initial events, i.e., breakage of the phosphodiester bonds. These phenomena are poorly understood in physicochemical terms; for instance, no conclusive work pertaining to the solubilization in acid media of nucleotides according to their size, conformation, or composition has been published. Moreover, it could well be that some intermediate products of hydrolysis can interfere with the enzymatic activity and modify its final expression, which is the only one recorded by the usual methods. Ease and Rapidity. One hundred determinations can be performed in about 6 hours. In other words, a complete set of chromatographic fractions can be tested for the presence or the absence of activity in a few hours, or the measurement of the enzymatic activity concentration can be performed on 20 different preparations in such a time, assuming that 5 points per kinetics for each preparation are enough to yield a fair estimate of this concentration. The only other practical method known for measuring the rate of DNA breakage at such a low concentration of nuclease is sucrose sedimentation. Two determinations a day are probably the best that can be performed with this technique.

Disadvantages of This Assay

Lack of Specificity. The changes of fluorescence intensities can be due to several causes not correlated to the opening of closed circles; for example, any cell extract can contain a relatively large amount of RNA, which binds EB and increases its quantum yield of fluorescence; if a RNase activity is present, the disappearance of RNA will induce a decrease of the fluorescence intensities which can deeply modify the kinetics due to the endodeoxyribonuclease. Another source of error is the existence of an exodeoxyribonucleolytic activity which will attack the circles as soon as they are opened by the endonuelease. To avoid any artifactual conclusion, control experiments must be run with a nicked or linear DNA replacing the closed circles. In this case, any increase of fluorescence intensities when the standard and the control assays are compared, can be attributed to an endonucleolytic activity. A treatment by RNase of any cell extract as previously described will destroy any RNA double-stranded structures able to bind EB. Scarcity and Fragility of the Substrate. The preparation of closed DNA circles in a satisfactory and reproducible yield was difficult to achieve until bacteriophage PM2 DNA was available.

[18]

DNA RESTRICTION ENZYME

269

In most preparations, there is a spontaneous opening of the closed circles at a slow rate (about half the X D N A circles opened in one month when kept at 0 ° in the dark). For this conservation, any reducing agent must be avoided. 1° Acknowledgments The authors wish to thank Mr. J. Paoletti and Miss E. Franque for their help. This work was supported by grants from the International Union against Cancer and from the Jane Coffin Memorial Funds, ~*V. C. Bode, J. Mol. Biol. 26, 125 (1967).

[ 18] D N A R e s t r i c t i o n E n z y m e f r o m g s c h e r i c h i a coli By ROBERTYUAN and MATTHEW MESELSON

Many strains of E. coli can recognize and degrade DNA from foreign E. coli strains. Whether a foreign DNA molecule is degraded depends on nonheritable properties imparted to it by the cell from which it is obtained. These are called host-controlled modifications. For example, the result of infecting E. coli strain K with phage X depends on the host in which the phages were last grown. Phages grown in bacteria possessing the modification character mk multiply with high efficiency, but phages grown in bacteria lacking mk do not. Instead, their DNA is quickly degraded on entering cells of strain K. The ability of strain K to degrade or "restrict" DNA from cells lacking mk is itself under genetic control, the responsible character being designated rk. There is evidence that the modification properties of a DNA molecule are determined by its pattern of secondary methylation. The simplest hypothesis for the biochemical basis of restriction is that each restriction character directs the formation of a nuclease specific for DNA lacking the corresponding modification. Such an endonuclease is present in strain K, and is specifically active against X DNA from strains lacking ink. The enzyme is called endonuclease R . K . 1

Assay Methods Principle. [SH] thymidine-labeled DNA is prepared from phage )~ grown on E. coli strain K, designated X. K DNA. 32p-labeled )~ DNA sensitive to endonuclease R ' K is prepared from a rk-mk- mutant of strain

1M. Meselson and R. Yuan, Nature (London) 217, 1110 (1968).