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Assays for Transcription Factor Activity 463

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Assay of Restriction Endonucleases 465

465

From:

Methods in Molecular Biology, vol. 148: DNA–Protein Interactions: Principles and Protocols, 2nd ed.

Edited by: T. Moss © Humana Press Inc., Totowa, NJ

Assay of Restriction Endonucleases

Using Oligonucleotides

Bernard A. Connolly, Hsiao-Hui Liu, Damian Parry,

Lisa E. Engler, Michael R. Kurpiewski, and Linda Jen-Jacobson

1. Introduction

Type II restriction endonucleases are familiar to most investigators in the

biological sciences as indispensable reagents for a variety of molecular

biology techniques. Usually, these dimeric enzymes cut double-stranded DNA

at defined, palindromic, sequences 4, 6, or 8 base pairs in length (1). Restric-

tion endonucleases have extremely high specificities, a key property that

underlies their applications in genetic engineering and which has attracted the

attention of scientists interested in sequence-specific DNA discrimination.

Considerable effort has been expended in trying to elucidate the mechanisms

that underlie specificity, often using the “structural perturbation” approach

(2,3). Here, an alteration is made to the protein by site-directed mutagen-

esis, or to the nucleic acid by substituting with natural base pairs, or with

synthetic base, sugar and phosphate analogs and the effects on binding and

cleavage observed. A useful measure of the perturbation can be obtained by

evaluation of the parameter

)

modified

/(k

)

unmodified

where “unmodified” refers to the native endonuclease and the natural DNA

target sequence and “modified” indicates that either the protein or the

nucleic acid has been changed in order to delete a selected interaction. The

parameter k

is the first-order rate constant obtained under single-turnover

conditions. Provided that the association of DNA and the essential cofactor

with the protein are rapid (see Note 1), k

measures the slowest step

following the assembly of the endonuclease–DNA–Mg

complex up to and

466 Connolly et al.

including the hydrolysis step. K

(the equilibrium dissociation constant) =

[E]

[D]

/[ED].*

The evaluation of (k

)

modified

/(k

)

unmodified

can be highly informative,

revealing how much a particular interaction contributes to the energetics of

DNA discrimination and whether the effect arises at substrate or transition state

binding (2,3). Examples include the use of oligonucleotides containing base

and phosphate analogs to probe DNA recognition by the EcoRI restriction

endonuclease (4,5). The underlying rationale of the “structural perturbation”

method have been treated comprehensively elsewhere (2) and are beyond the

scope of this chapter. Similarly, methodology for the alteration of proteins by

site-directed mutagenesis and the preparation and uses of oligonucleotides con-

taining modified bases, sugars, and phosphates will not be discussed. Rather,

this chapter will concentrate on the practical aspects of k

and K

measurement.

In order to measure k

, the oligonucleotide is labeled with

P, most com-

monly at the 5' terminus using γ-[

P]-ATP and polynucleotide kinase. Alter-

natively, labeling at the 3' end with α-[

P]-ddNTP and terminal transferase

can be used. Restriction endonucleases have an absolute requirement for Mg

enabling the reaction to be initiated in a number of ways: usually the addition

of Mg

to a premixed solution of an enzyme plus oligonucleotide or the addi-

tion of an enzyme to a solution containing the oligonucleotide and Mg

(see

Note 1). At various times aliquots are removed and the reaction quenched. The

substrate and product DNA are separated using denaturing polyacrylamide gel

electrophoresis (PAGE) and quantitated by phosphorimaging. The data

obtained are fitted to appropriate equations to give k

values. Several consider-

ations need to be taken into account:

• Measurement of a rate constant under single-turnover conditions requires all the

substrate to be bound to the enzyme at the start of the reaction. This necessitates

(a) [E]

> [D]

and (b) [D]

>> K

(see Note 2).

• When a wild type restriction endonuclease is used to cut its natural DNA target,

both strands of the duplex are usually cut at the same rate. However, introducing

a modification into only one of the DNA strands often results in the individual

strands being cut at different rates. In these cases cleavage is most simply and

accurately measured if the two substrate strands and the two labeled product

strands can be separated, as shown in Fig. 1.

• Measurement of DNA cleavage by restriction endonucleases, under single-turn-

over conditions, approaches the limit of manual manipulation. In some cases, it

has been possible to mix the enzyme with its substrate and withdraw aliquots by

*Throughout this chapter, E = endonuclease; D = DNA; ED = enzyme–DNA complex. The

subscripts t and f denote the total and free amounts of E and D present (i.e., [E]

= [E]

– [ED];

[D]

= [D]

– [ED]). Several investigators use K

(= 1/K

) rather then K

. Both terms are accept-

able, and in some instances in this chapter K

has been used.

Assay of Restriction Endonucleases 467

hand. In others, the reaction is too fast to be evaluated in this manner and a rapid-

mixing quenched flow apparatus must be used. Alteration to the endonuclease or

the DNA often cause a reduction in the single-turnover rate constant and it is

often possible to use manual methods in these cases.

The most suitable method for K

determination depends critically on its

magnitude and hence the stability of the protein–DNA complex (see Note 3).

Several approaches for the measurement of K

are presented, enabling equilib-

rium constant evaluation under conditions that range from very tight (K

≈ pM)

to very weak (K

≈ mM) binding. When commencing experiments, the K

will

not be known and good practice requires an initial estimate, followed by an

accurate evaluation using the optimal approach. Modification to the endonu-

clease or the DNA often weakens their interaction, sometimes by a consider-

able amount. Therefore, the method used in the “natural” case may not be the

best when alterations to the macromolecules are present. Whatever method is

used, it is important to prevent DNA hydrolysis and this is most simply

achieved by omission of Mg

and addition of EDTA.

Two commonly used methods for K

determination are filter binding and

gel retardation (electrophoretic mobility shift assay [EMSA]). Both are dis-

cussed elsewhere in this volume (see Chapters 1 and 2) and this chapter

Fig. 1. Oligonucleotides that have been used to measure single turnovers with the

EcoRV and EcoRI restriction endonucleases (recognition sequence shown with line,

cleavage sites with arrows). With EcoRV, both the substrate strands are different

lengths. In the case of EcoRI, the two substrate strands can be separated by denaturing

gel electrophoresis, despite having the same length, as the top strand is dG rich and the

bottom dC rich. dC-rich strands migrate faster than dG (dA and T base-pairs affect

migration much less). With EcoRV 3' labeling (*ddA) was used, and with EcoRI, 5'

labeling (*P) was used. The offset recognition sites gives products of different sizes.

The two arrangements ensure the separation of both substrate and both labelled prod-

uct strands and so allow independent measure of the rate of cleavage of the two strands.

Only the labeled products are shown. For both enzymes the “top” strands are written

in the 5' → 3' direction and the “bottom” are written in the 3' → 5' direction.

468 Connolly et al.

emphasizes their application to restriction endonucleases and also the use of

competition titration. Most often, a

P-labeled oligonucleotide is incubated

with increasing amounts of restriction enzyme to produce a protein–DNA com-

plex. The free and protein-bound DNA are then separated and the relative

amounts in each pool determined. This allows the construction of a binding

isotherm and K

evaluation. Data analysis is simplified when [E]

≈ [E]

and

experimentation is usually carried out under these conditions (see Note 4). It is

also useful to carry out a “reverse” titration: incubating a fixed amount of

endonuclease with increasing quantities of

P-labeled oligonucleotide.

Titrations with a fixed [DNA] may give rise to shifts in the oligomeric state of

the protein because of its increasing concentration, which can produce biphasic

isotherms or curves that do not show asymptotic behavour. This arises due to

the coupled reactions E

+ E

→ E

(where E

is the active form) and is

particularly relevant to modified substrates that show weak binding; requiring

titration with concentrations of protein that may exceed the K

for the coupled

dimer–tetramer equilibrium. If both procedures give identical K

values, one

has confidence that the coupled reaction can be ruled out as an obfuscating

factor. In addition, the combination of the two approaches permits precise

determination of the number of active protein molecules, providing that the

DNA concentration is accurately known.

Filter binding and gel retardation differ in the manner used to separate the

free DNA and the protein–DNA complex. In filter binding, separation is

achieved using a nitrocellulose filter that retains proteins, and hence protein–

DNA complexes, but allows the passage of free DNA. Some proteins are poorly

bound by nitrocellulose filters although this difficulty can sometimes be cir-

cumvented by using a different filter material (e.g., pure nitrocellulose rather

than mixed-ester filters). Gel retardation uses non denaturing PAGE; free DNA

migrates more rapidly than the protein–DNA complex giving, under ideal con-

ditions, two well-resolved bands. Both approaches are sensitive (can be used at

very low [DNA] to measure tight binding), experimentally very simple to carry

out, and do not require specialized equipment.

Whether filter-binding or gel-shift methods are chosen, the direct titration

protocol is not recommended for modified substrates that show very weak bind-

ing (i.e., K

> 10

–7

M), because the lifetime of the complexes (see Note 3) is

always shorter than the time required either for filtration or for entry into the

gel (see Note 5). This may result in partial dissociation of the endonuclease–

DNA complex during the measurement (i.e., a “nonequilibrium” situation).

For weak binding, a competition titration (6) protocol should be used. Here, a

fixed quantity of an endonuclease and a

P-labeled oligonucleotide (most often

containing an unmodified recognition sequence for the endonuclease under

study) are allowed to form a complex. Progressively increasing amounts of a

Assay of Restriction Endonucleases 469

nonradioactive competitor DNA (e.g., a variant oligonucleotide in which there

has been a base-analog or natural base-pair substitution) are added. The unla-

beled DNA molecules compete with the

P-labeled oligonucleotide for the

DNA-binding site on the protein and so leads to a displacement of the radioac-

tive probe. It is good practice to compare some of the K

values obtained by

the direct method with those obtained by competition titration, both to test

whether equilibrium is significantly perturbed in the direct method and to pro-

vide confidence that all binding reactions have achieved equilibrium in the

more indirect competition method. Thus, one should include in the experimen-

tal set of modified oligonucleotides at least one “internal control” for which

both direct and competition titrations can be performed (see Note 6).

Equilibrium constants can also be determined using fluorescence anisotropy

(7,8) with oligonucleotides labeled with hexachlorofluorescein. When a

fluorophore is excited with plane polarized light, the extent to which the emitted

light becomes depolarized depends on the rate at which the fluorophore tumbles

(which, in turn, depends on molecular weight) and also the lifetime of the fluo-

rescence excited state (see Note 7). When a protein binds to an fluorescent

labeled oligonucleotide, the mass associated with the fluorophore, and there-

fore the fluorescence anisotropy, is increased, allowing K

determination.

Hexachlorofluorescein is commercially available as a phosphoramidite suit-

able for automated DNA synthesis, enabling its attachment to the 5' terminus

of an oligonucleotide via a six-carbon-chain linker (Fig. 2). The flexible linker

gives the probe a degree of motion independent of the dynamics of the DNA or

the protein–DNA complex. However, although the probe is not rigidly attached

to the DNA, it has enough movement coupled to the DNA, together with

an appropriate fluorescence lifetime of about 3 ns for the probe attached to a

21-mer (9), to make it sensitive to protein binding (see Note 7).

The minimum concentration of hexachlorofluorescein that can be easily

measured is about 1 nM and this limits the technique to measuring K

values

of approx 1 nM and above. It has been used to measure K

values that approach

Fig. 2. The structure of hexachlorofluorescein and its linkage to the 5'-phosphate of

an oligonucleotide.

470 Connolly et al.

1 µM. Thus, fluorescence anisotropy is much less sensitive than filter binding

or gel retardation, which rely on the detection of

P. The biggest advantage of

this method is that it is a strictly equilibrium approach and does not suffer from

problems caused by protein–DNA dissociation. Drawbacks include the require-

ment for expensive equipment and inability to measure tight binding (K

< 1 nM).

2. Materials

2.1. Oligodeoxynucleotides and Restriction Endonucleases

1. [

P]-labeled oligodeoxynucleotides. These should be of high specific activity,

prepared using either γ-[

P]-ATP (3000–6000 Ci/mmol)/polynucleotide kinase

(5' labeling) or α-[

P]-ddATP(>5000 Ci/mmol)/terminal transferase (3'-label-

ing) and purified by standard procedures (10). Duplex oligodeoxynucleotides are

prepared by heating equimolar amounts of each strand to 95°C in buffer with the

same pH value and salt concentration to be used in k

or K

measurement and

cooling slowly to room temperature. For k

determination, both strands must be

labeled; for K

evaluation, only one strand needs to be labeled.

2. Oligodeoxynucleotides containing hexachlorofluorescein at their 5' termini.

These can be prepared by chemical synthesis using commercially available

hexachlorofluorescein–phosphoramidite (Glen Research, Sterling, VA;

Cruachem Ltd., Glasgow, Scotland) and purified by reverse-phase high-perfor-

mance liquid chromatography (HPLC) (9). Duplexes, only one strand of which

needs to contain the fluorophore, are prepared as in item 1.

3. Restriction endonucleases under study. In this case, EcoRI, EcoRV, and BamHI

purified from overproducing Escherichia coli strains.

2.2. k

Determination

1. Vertical slab gel apparatus (140 × 160 k 0.75 mm) (e.g., Hoefer SE600 or equiva-

lent) and power pack with ≈200 V direct current output.

2. Denaturing polyacrylamide gel. Prepared from 16% acrylamide (acrylamide/bis-

acrylamide, 19/1) in 0.089 M Tris–borate, pH 8.0, containing 8 M urea and 1 mM

EDTA and polymerized with 0.05% ammonium persulfate (added from a freshly pre-

pared 10% aqueous solution) and 1% TEMED (N,N,N'N'-tetramethylethylene

diammine). The gel should be prerun at a constant power of 30 W, for 1 h, prior to use.

3. Gel running buffer: 0.089 M Tris–borate, pH 8.0, containing 1 mM EDTA.

4. Hydrolysis buffer (made up at 2X the final concentration, freshly prepared and

stored at 4°C): in the examples given using EcoRV endonuclease; 20 mM HEPES,

pH 7.5, 200 mM NaCl, and 20 mM MgCl

(see Note 8).

5. Enzyme dilution buffer: Incubation buffer plus 5% (v/v) glycerol (see Note 8).

6. Stop solution: 0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA, 2.5 M urea, 10% (w/v)

sucrose, and 125 mg/mL (each) bromophenol blue and xylene cyanol FF.

7. Vacuum bag sealer and plastic sheets.

8. Phosphorimager (e.g., Fuji BAS-150) and phosphorimager screen (e.g., Fuji

BAS-MP) (or equivalent).

Assay of Restriction Endonucleases 471

9. Quenched flow apparatus (e.g., Hi-Tech RQF–63 [Hi-Tech Scientific, Salisbury,

UK]) or equivalent.

2.3. K

Determination (Filter Binding)

1. Vacuum filtration manifold (e.g., Millipore 1225 Sampling Vacuum Manifold,

Bedford, MA).

2. Mixed ester nitrocellulose membrane filters (e.g., Gelman GN6, Millipore

HAWP02500, Schleicher & Schuell ME25) or pure nitrocellulose membrane fil-

ters (e.g., Schleicher & Schuell, type BA85), 25 mm disks, 0.45-µm pore size.

3. Enzyme dilution buffer: Binding buffer plus 5% glycerol and 0.2 M or 0.6 M

NaCl (final concentrations), pH 7.4 (see Note 8).

4. Filter buffer (stored at 4°C): 10 mM BTP (bis-Tris propane), 1 mM EDTA, 0.02%

NaN

at desired pH (e.g., pH 7.4) and desired salt concentration (e.g., 0.2 M

NaCl) (see Note 8).

5. Binding buffer (stored at 4°C): Filter buffer plus 0.1 mg/mL bovine serum albu-

min and 50 µM dithiothreitol (DTT).

6. Polyethylene liquid-scintillation vials (6 mL capacity).

7. Liquid-scintillation fluid (e.g., Scintisafe 30%, Fisher Biotech, Pittsburgh, PA).

8. Liquid-scintillation counter (e.g., Packard model 1600; Meridan, CT).

2.4. K

Determination (Gel Retardation)

1. Vertical slab gel apparatus (140 × 160 × 0.75 mm) (e.g., Hoefer SE600 or equiva-

lent) and power pack with ≈200 V direct current output.

2. 10% Nondenaturing polyacrylamide gel (acrylamide/bis-acrylamide, 37.5/1) in

0.089 M Tris–borate, pH 8.0, and 1 mM EDTA and polymerized with 0.05%

ammonium persulfate (added from a freshly prepared 10% aqueous solution) and

1% TEMED. The gel should be prerun at a constant power of 20 W with gel

running buffer and the wells rinsed with this buffer, prior to use.

3. Gel running buffer: Usually 0.089 M Tris–borate, pH 8.0, containing 1 mM

EDTA. However, the running buffer can, to a limited extent, be varied to produce

a better match with the binding buffer (see Note 9).

4. Binding buffers and enzyme dilution buffers: As in Subheading 2.3., items 3 and 5

except that the binding buffer should additionally contain 3% (v/v) glycerol (see Note 10).

5. Bromophenol blue solution: 0.25% (w/v) in 30% (v/v) glycerol.

6. Vacuum bag sealer and plastic sheets.

7. Phosphorimager e.g., Fuji BAS–150 and phosphorimager screen (e.g., Fuji

BAS-MP) (or equivalent).

2.5. K

Determination (Fluorescence Anisotropy)

1. Fluorimeter, with thermostated cuvet compartment, capable of measuring fluo-

rescence anisotropy e.g., Aminco SLM-8100.

2. 3-mm-thick 570-nm longpass filter, (OG-570, Schott Glaswerke).

3. 0.5 mL semimicro quartz fluorescence cuvets (excitation and emission

pathlengths 5 mm).

472 Connolly et al.

4. Fluorescence binding buffer and enzyme dilution buffer: In the examples given

with the EcoRV endonuclease, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM

DTT, 1 mM EDTA, and 0.1 mg/mL acetylated bovine serum albumin. The fluo-

rescence binding buffer should be prepared using the best quality reagents and

HPLC-grade water and both degassed and passed through 0.22-µm filters (e.g.,

Millex-GV

, Millipore) prior to use.

2.6. Data Analysis

Software capable of fitting reaction data to single and multiple exponential

decay(s) and binding data to single-site binding isotherms using nonlinear

regression analysis. The software used for binding analysis should be able to

deal with direct titrations when the simplifying assumption [E]

= [E]

cannot

be made (see Note 4) and competitive titrations. Three packages described in

this chapter are GraFit (11), Scientist (12), and SigmaPlot (13), but any soft-

ware with a similar capability is perfectly acceptable.

3. Methods

3.1. Determination of k

3.1.1. Slow Hydrolysis (t

1/2

15 s or More

≈

Values Slower

Than 3 min

–1

; K

Between 2 and 40 n

)

(e.g., with Mutant

Eco

RV or Altered Oligodeoxynucleotides)

1. In a plastic microcentrifuge tube, prepare 11 µL of a solution containing 1.5 µM

of radiolabeled DNA duplex in hydrolysis buffer (in this case for the EcoRV

endonuclease, 10 mM HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl

) (see

Note 8). Keep on ice.

2. Withdraw 1 µL of the above solution and add to 10 µL of stop solution (item 6 of

Subheading 2.2.). This serves as a zero time-point.

3. Prepare 5 µL of a solution containing 30 µM of EcoRV endonuclease in 10 mM

HEPES, pH 7.5, 100 mM NaCl, and 10 mM MgCl

. Keep on ice.

4. Incubate the solutions prepared in steps 1 and 3 at 25°C for 10 min.

5. Mix the two solutions to give a final oligonucleotide concentration of 1 µM and a

final endonuclease concentration of 10 µM (see Notes 1 and 2). Vortex briefly to

ensure thorough mixing and incubate at 25°C.

6. Withdraw 1-µL aliquots at times up to 25 h (the exact time range to be used must

be found by trial and error and will depend on the particular mutant enzyme

and/or altered oligodeoxynucleotide under study) and quench the reaction by

the addition to 10 µL of stop solution (item 6 of Subheading 2.2.). Store the

quenched samples on ice.

7. Run the samples on 16% denaturing polyacrylamide gels (item 2 of Subheading

2.2.) until the bromophenol blue dye marker reaches the bottom of the gel.

8. Remove the gel from the electrophoresis apparatus and seal in a plastic bag using

a vacuum bag sealer.