Purification and electrophoretic assay of T4-induced polynucleotide ligase for the in vitro construction of recombinant DNA molecules

ANALYTICAL

BIOCHEMISTRY

75,

545-554 (1976)

Purification and Electrophoretic Assay of Tdlnduced Polynucleotide Ligase for the in Vitro Construction of Recombinant DNA Molecules STEPHEN Department

of Chemistry.

University

K. MOORE AND ERIC JAMES of South

Carolina,

Columbia,

South

Carolina

29208

Received February 25. 1976; accepted June 8, 1976 A facile method for the determination of bacteriophage TCinduced polynucleotide ligase joining activity is described. The assay is based on the ability of polynucleotide ligase to join the cohesive termini of bacteriophage A DNA covalently. The observance of this activity is greatly facilitated if A DNA is previously cleaved with the restriction endonuclease EcoRI and the reaction products subsequently analyzed by electrophoresis in ethidium bromide-agarose gel. A purification scheme is presented which offers a reduction in the number of steps required to purify polynucleotide ligase compared to a previously published procedure and yields an enzyme preparation which is suitable for use in in vitro construction of recombinant DNA molecules.

There is considerable interest in the development of studies involving the in vitro construction of recombinant DNA molecules (1); these studies require the use of relatively large quantities of polynucleotide ligase which is commercially expensive and difficult to purify using established protocols. In order to simplify the purification of this enzyme, a rapid and facile assay which allows the determination of enzyme activity of multiple samples is needed. Assay procedures previously utilized (2-6) involve the preparation of radioisotope-labeled DNA substrates requiring the prior purification of several other enzymes or, alternatively, the laborious analysis of reaction products by means of density gradient centrifugation. Although the ATP-PPi exchange assay (3) is equally as simple as the assay presented herein, it does not provide a method for the determination of the actual nucleic acid joining capacity of an enzyme preparation of polynucleotide ligase. We describe an assay which is based on a previous observantion that polynucleotide ligase converts hydrogen-bonded circles of A DNA to a covalently closed form (2,7). We have found that the determination of polynucleotide ligase activity is greatly facilitated if A DNA is previously cleaved with the restriction endonuclease EcoRI and the reaction products subsequently analyzed by electrophoresis in ethidium bromide-agarose gels. Eco RI is generally required for the construction of recombinant DNA molecules and is the only enzyme needed 54s Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved

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AND

JAMES

for preparation of the DNA substrate. The purification protocol for polynucleotide ligase described in this work is based on that described by Weiss et al. (3) and our observation (Moore and James, unpublished results) that the bulk of the exonuclease activity present in crude cellular extracts of Escherichia coli B elutes from phosphocellulose at low ionic strength (co.25 M KCl) whereas polynucleotide ligase elutes at approximately 0.30-0.36 M KCl. The utilization of gradient elution from phosphocellulose as the first chromatographic step in the purification protocol permits the assay of polynucleotide ligase without the polynucleotide joining reaction being masked by exonuclease activity. MATERIALS

AND METHODS

Reagents. Agarose, dithiothreitol, glutathione (reduced form), and bovine plasma albumin were purchased from Sigma Chemical Co. Dipotassium ATP (grade A) was purchased from Calbiochem. Ammonium sulfate (enzyme grade) was purchased from Schwarz/Mann. All other chemicals were of reagent grade purchased from commercial sources. Strains and bacteriophage. RY-13 (E. cofi B carrying the RTF-1 drug resistance transfer factor) was the gift of B. Allet. Bacteriophage T4 and E. coli B/5 for the preparation of polynucleotide ligase and E. cofi JC411 thy(Co1 El) for the preparation of the colicin El factor were the gift of B. Weisblum. An E. coEi lysogen of AcZ857susS7 was the gift of N. Kelker. Enzymes. The specific endonuclease EcoRI was purified by the method of B. Allet (personal communication). The procedure involves phosphocellulose, hydroxyapatite, and DEAE-cellulose chromatography. TCinduced polynucleotide ligase was prepared by a modification of the procedure of Weiss et al. (3) which greatly facilitates the monitoring of enzyme activity. Bacteriophage T4 was propagated as previously described (8). Growth of cells and phage infection was performed as described (3). The culture was harvested by passing it rapidly through stainless-steel coils immersed in an ice bath followed by centrifugation at 60008 for 7 min. Cells were stored at -70°C. All subsequent operations were carried out at 4°C and all buffers contained 7mM /3-mercaptoethanol unless otherwise indicated. Frozen cells (65 g) were suspended in 450 ml of Buffer A (10 mM Tris-HCI, pH 7.6, containing 1 mM glutathione in place of @-mercaptoethanol) and disrupted by sonication for 10 min (20 bursts of 30 set each) with a Branson Model W185 sonic oscillator at maximum power setting. Cellular debris was removed by centrifugation for 60 min at 100,000 g. Nucleic acid was removed by precipitation with streptomycin sulfate as previously described (9). The supernatant fluid was carefully decanted and tested for the completeness of nucleic acid precipitation by the addition of a drop of streptomycin sulfate solution. Crystalline ammonium sulfate was added slowly with stirring to 50% saturation and the

POLYNUCLEOTIDE

LIGASE

TABLE PURIFICATION

1

OF POLYNUCLEOTIDE

Volume

547

ASSAY

[Protein]

LIGASE

Total protein

(mg)

Activity (units X 1O-6)

Fraction

(ml)

O-ndml)

I. Crude extract 11. Ammonium sulfate (O-50%) III. Phosphocellulose eluate IV. DEAE-cellulose eluate V. Glycerol concentrate

400

26.5

IO.600

-

100

4.6

460

-

255

0.15

38

3.6

250 68

0.045 0.16

11.3 10.9

1.9 1.7

mixture stirred for an additional 30 min. After centrifugation at 15,OOOg for 20 min, the pellet was dissolved in lOOm1 of Buffer A (Fraction II), and dialyzed 18 hr against 3 x 1 liter of 10 mrvr potassium phosphate buffer containing 0.1 M KCl, pH 7.6 (Buffer B). The preparation was applied to a 2.5 x 50-cm phosphocellulose column (Whatman, P-11) previously equilibrated with Buffer B. The column was washed with 1 liter of Buffer B and a linear gradient (total volume, 2 liters) of 0.1-0.8 M KC1 in 10 mM potassium phosphate buffer, pH 7.6, was applied. Column fractions were assayed as described below. The active fractions (>lOOO units/ml), emerging between 0.30 and 0.36 M in KCl, were pooled (Fraction III) and dialyzed 18 hr against 3 x 2 liters of 15 mM Tris-HCI containing0.05 M KCI, pH 7.6 (Buffer C). The preparation was applied to a 2.5 x 50-cm DEAE-cellulose column (Whatman, DE-52) previously equilibrated in Buffer C, washed with 1 liter of Buffer C, and eluted with a linear gradient (total volume 2 liters) of 0.05-0.3 M KC1 in 15 mM Tris-HCl, pH 7.6. The active fractions, emerging between 0.12 and 0.15 M KC1 were pooled (Fraction IV), concentrated by dialysis against Buffer C containing 5% glycerol, and stored at -20°C. Preparation of DNA substrate. Preparation of AcI857susS7 bacteriophage and subsequent isolation of the bacteriophage DNA has been described (IO). The DNA was digested with the endonuclease EcoRI as previously described (11). The endonuclease EcoRI fragments of A DNA were extracted once with phenol saturated with 10 mM Na,-EDTA, pH 8.0. and dialyzed exhaustively against 10 mM Tris-HCl, pH 7.6. In order to promote the joining of A fragments 1 and 6 (Fig. 2) via hydrogen bonding of their A cohesive termini, an aliquot of the fragment preparation was adjusted to 10 mM in MgCl,, incubated at 50°C for 3 hr. and allowed to cool slowly. Annealing in the presence of Mg”+ (12) allows rapid joining of A cohesive termini without the use of high Na+ concentrations. Assay for polynucleotide ligase. The assay described herein measures

548

MOORE AND JAMES

abed

FIG. 1. Demonstration of the lack of interfering activities in the final polynucleotide ligase preparation (Fraction V). Gel a, native A DNA was incubated in a reaction mixture minus ATP for 3 hr at 37°C with 100 enzyme units. the reaction terminated, and the product of the reaction subjected to ethidium bromide-agarose gel electrophoresis. Gel b. DNA substrate. Gel c, DNA substrate after incubation on ice in the standard reaction mixture with 100 enzyme units for 18 hr. Gel d, DNA substrate incubated with 100 enzyme units for 3 hr at 37°C then transferred to ice for an additional 8 hr. Agarose-gel electrophoresis was performed according to the method of Sharp et al. (9). In all cases, electrophoresis was carried out from top (cathode) to bottom (anode). and the amount of DNA applied to each gel was 0.5 pg.

the conversion of hydrogen-bonded endonucleaseEcoR1 fragments 1 and 6 of A DNA to a nonheat-denaturable (covalently closed) form. The standard reaction mixture contained in a total volume of 25 ~1: 2.5 ,ug of endonucleaseEcoRIfragmentsofhDNA,50m~Tris-HC1,pH7.6, Lomb

POLYNUCLEOTIDE

LIGASE

ASSAY

549

abcdefqhi

FIG. 2. Electrophoretic profiles of A&oRI fragments incubated with varying amounts of polynuch :otide ligase. Polynucleotide ligase (Fraction V) was titrated againstEcoRI-digested A DNA i,n which the terminal fragments (fragments 1 and 6) were previously joined by hydrogen bonding. The numbering system is such that fragment 1 exhibits the lowest electrophl aretic mobility( 19). Gel a, DNA substrate;gefs b through iO.O,O. I. 0.3.0.5.0.7. 1.0, 1.5, and Z!.O units of polynucleotide ligase were present in the standard reaction mixture, respective :ly.

dithiothreitol, 0.07 mM ATP, and varying amounts of enzyme diluted in 50 mM Tris-HCI, pH 7.6, containing 500 CLg/ml of bovine plasma albumin. The mixture was incubated for 30 min at 37°C. The reaction was terminated by the addition of four volumes of 5 mM Nk-EDTA, pH 8.0. The mixture was heated to 65°C for 10 min and rapidly cooled to 0°C in order to separate noncovalently linked fragments. Sucrose and bromophenol blue (final

550

dOORE AND JAMES

---6

-1 0

I

2

3

4

5

IO

Migration, cm FIG. 3. Densitometric scans of electrophoretically separated products of ligation. The fluorescence intensity of ethidium bromide-stained DNA bands were recorded by photography, and the optical density profile of negatives was obtained as described in Materials and Methods. Polynucleotide ligase was present in the standard reaction mixture in the following amounts: Scan (a), 0.0 unit; (b), 0.5 unit; (c), 1.0 unit: (d) 2.0 units. Vertical bar indicates 1 optical density unit.

concentrations 10 and 0.02% (w/v) , respectively) were added to a 25-/d aliquot of the above preparation. Samples were layered onto IO-cm 0.8% agarose-ethidium bromide gels and electrophoresis performed as previously described (9). DNA band patterns were visualized by illumination of the gels with short-wave light. One unit of enzyme is defined as that amount which covalently joins the cohesive ends present in 1 pg of A DNA (3.9 x lOlo termini) under the standard assay conditions. Visually, this corresponds to that amount of enzyme which renders A fragment 6 undetectable in the gel pattern (Fig. 2). The fluorescence of the ethidium bromide-stained DNA bands was

POLYNUCLEOTIDE

0.0

0.2

0.4

Units

0.6

LIGASE

0.6

1.0

1.2

polynucleotide

551

ASSAY

1.4

1.6

l.6

2.0

ligase

FIG. 4. Relationship of the extent of the ligation reaction and the amount of polynucleotide ligase present in the standard reaction mixture. The percentage of covalently closed A cohesive termini was determined from the relative amount of A fragment 6 in the chromatogram.

recorded on Kodak Tri-X panchromatic film (ASA 400). Conditions for photography were a 30- to 90-set exposure, f4.7, and a Tiffen 25A filter. Gels containing various known amounts of electrophoretically separated endonuclease EcoRI fragments of A DNA were included in each exposure in order to allow for subsequent quantitation of the fluorescence. The reported molecular weight values (13) of these fragments were used to calculate the amount of nucleic acid present in each band. Optical density scans of the negatives were obtained with a Gilford Model 250 spectrophotometer equipped with a Model 2520 linear transport device. Since the majority of optical density measurements were found to be in the linear range of the film, the appropriate portions of the chromatogram were cut out and weighed to determine the relative ratio of peak areas. A plot of peak area against the amount of DNA present in each band of the standards was used to determine the percentage of A fragment 6 which was covalently joined to A fragment 1 by polynucleotide ligase. RESULTS AND DISCUSSION

The procedure outlined in Materials and Methods for the purification of polynucleotide ligase offers a reduction in the number of steps required to purify the enzyme compared to the method of Weiss et al. (3) The major difference in procedure is a change in the order of chromatographic protocols used and omission of a DEAE-cellulose fractionation step. An accurate determination of polynucleotide ligase activity prior to elution from phosphocellulose was precluded by extensive degradation of

552

MOORE AND JAMES

the DNA substrate due to the presence of large amounts of exonuclease. The joining assay described herein, was however, possible for the remaining steps and the results are presented in Table 1. Polynucleotide ligase purified through the first chromatographic step (Fraction III) was found to be slightly contaminated with exonuclease as evidenced by a lack of sharpness of the DNA bands when the reaction products of this fraction were subjected to electrophoresis. The final enzyme preparation (Fraction V), at least under conditions suitable for joining of polynucleotides, contains negligible exonuclease, endonuclease, or phosphatase activity or activity of other inhibitors of ligation. There was no detectable degradation of 1 pg of A DNA after incubation for 3 hr in the presence of 100 enzyme units of polynucleotide ligase (Fraction V) since the product of incubation migrated in agarose gel as a single sharp band corresponding to native A DNA (Fig. 1, gel a; ATP was omitted from the reaction mixture to prevent concatemer formation). Endonuclease EcoRI fragments of A DNA (1 pg) were incubated for 3 hr with 100 enzyme units of polynucleotide (Fraction V) ligase and subjected to electrophoresis. Only those fragments which correspond to known endonuclease EcoRI cleavage sites were found. Furthermore, when 1 pg of Co1 El DNA (14) was incubated under the same conditions, the product of the reaction migrated in agarose gel coincident with native (circular) Co1 El DNA (data not presented). These data indicate the absence of significant exonuclease or endonuclease contamination of the polynucleotide ligase preparation. Incubation of 1 pg of endonuclease EcoRI A fragments with 100 enzyme units of polynucleotide ligase (Fraction V) at 37°C followed by incubation for 18 hr on ice resulted in extensive joining of the DNA fragments (Fig. 1, gel d). This indicates that the endonuclease EcoRI-generated termini were also undamaged by incubation with 100 times the normal amount of Fraction V polynucleotide ligase . Figure 2 shows the electrophoretic profiles of the endonuclease EcoRI fragments of A DNA after incubation with increasing amounts of polynucleotide ligase under standard assay conditions. In order to facilitate its interpretation, optical density scans are presented in Fig. 3. Progress of the reaction is indicated by a decrease in the intensity of bands corresponding to fragments 1 and 6 with a concomitant increase in a band due to the covalently joined fragments. Figure 4 indicates that the conversion of hydrogen-bonded A cohesive termini is proportional to enzyme concentration up to the approach of the completion of the reaction (ca. 1 unit). The data in Figs. 3 and 4 also indicate that the midpoint (0.5 unit) of a titration experiment can be identified visually as the point at which the intensity of the bands corresponding to fragments 1 + 6 and fragment 1 is roughly equal. The present work demonstrates that a high

POLYNLJCLEOTIDE

LIGASE

ASSAY

553

percentage (at least 95%) of the original hydrogen-bonded cohesive termini are converted in the presence of excess polynucleotide ligase to the covalently closed form. Previous studies (2,3,7,15), involving several different assay procedures, have reported lower values ranging from 35 to 70% conversion. It has been previously shown that DNA fragments bearing EcoRIgenerated termini (T, - S-6°C) can be joined by polynucleotide ligase to form circular or concatemeric structures (16). These fragments are terminated by the short (5’) PA-A-T-T- sequence (17). The hydrogen-bonded termini of bacteriophage A DNA, however, consist of a 1Zbase sequence (18) and exhibit aconsiderably higher melting point (12). We have observed that at 37°C only the endonuclease Eco RI fragments 1 and 6 of A DNA. which each bear one X cohesive end, can be covalently joined by polynucleotide ligase, thus greatly facilitating the quantitation of enzymatic activity. The assay procedure and the protocol for the purification of polynucleotide ligase described in this work offer a facile method for the preparation of this important enzyme which should prove to be of value to workers involved in studies concerning the in vitro construction of recombinant DNA molecules. ACKNOWLEDGMENTS We wish to thank Drs. B. Weisblum and N. Kelker for advice and the gift of strains, Dr. S. Goode for advice on fluorescence quantitation, and Dr. R. Dunlap for a helpful discussion of enzyme kinetics. This work was supported by the American Cancer Society (Grant No. VC 130 and VC 131A) and the National Cancer Institute of the U.S. Public Health Service (Grant No. CA 17830-01).

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12. Wang, J. C., and Davidson, N. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 409-415. 13. Thomas, M., and Davis, R. W. (1975)J. Mol. Biol. 91, 315-328. 14. Clewell, D. B. (1972) .I. Bacterial. 110, 667-676. 15. Zimmerman, S. B., Little, J. W., Oshinsky, C. K., and Gellert, M. (1967) Proc. Nat. Acad. Sci. USA 57, 1841-1848. 16. Dugaiczyk, A., Boyer, H. W., and Goodman, H. M. (1975)J. Mol. Biol. 96, 171-184. 17. Hedgpeth, J., Goodman, H. M., and Boyer, H. W. (1972)Proc. Nat. Acnd. Sci. USA 69, 3448-3452. 18. Wu, R., and Taylor, E. (1971)3. Mol. Biol. 57, 491-511. 19. Helling, R. B., Goodman, H. M., and Boyer, H. W. (1974) J. Virol. 14, 1235-1244.