THE JOURI\‘AL OF BIOLOGKXL CIIEMI~TRY

Vol. 243, No. 17, Issue of September 10, pp. 4543-4555, 1968
Printed in U.S.A.

Enzymatic Breakage and Joining of eoxyribouucleic Acid

VI. E’URTHER PURIE’ICATION AND PROPERTIES OF POLYNCCLEOTIDE LIGASE FROM EXCHERICHIA
COLI ISFECTED WITH BACTERIOPHAGE T4*

(Received for pLtblicat,ion, May 2, 1968)

UEI~NAKI) WEISS,$ ALAIN JACQUE~\IIN-SABLON,! THEOI)OHE It. LIVE,~ GEORGE C. FAREED, AND CHARLES C.
~~KXAR~SON
From the Depadment of Biological Chenaistry, Harvad Medical School, Boston, Massachusetts0.2115

SUMMARY
Polynucleotide ligase has been purified lOOO-fold from single strand break in DNA, from attack by bacterial alkaline
extracts of Escherichia coli infected with bacteriophage T4. phosphatase. The enzyme purification procedure has been
The enzyme catalyzes (a) the repair of phosphodiester bond redesigned to eliminate the necessity for enzyme assays
scissions introduced into the strands of bihelical DNA by during the early fractionation steps and to permit the use of
pancreatic DNase and (b) the conversion of hydrogen-bonded a simple assay, based on ATP-PPi exchange, during the later
circular duplexes or concatemers of X bacteriophage DNA to stages of purification.
covalently bonded circular molecules or concatemers, respec- The ligase reaction has a specific requirement for a bihelical
tively. The DNA products of these reactions have been DNA substrate and for ATP, and it results in the formation
characterized by sedimentation and end group analysis. of phosphodiester bonds, AMP, and pyrophosphate. The
The ligase does not catalyze the end to end joining of T7 K, for DNA containing single strand breaks is 1.5 X 10Vg M
bacteriophage DNA. expressed as the concentration of internal phosphomono-
The standard assay for ligase activity measures the pro- esters. The K, for ATP is 1.4 X 10-j M, and dATP is a
tection of a 32P-labeled 5’-phosphoryl group, located at a competitive inhibitor of the enzyme (Ki of 3.5 X lop5 M).

3’- - - , , , , --- 5 3’ - - - , , , , --- 5’

A T G T ATGT
TACA + ATP - T A C A + AMP + PPi

51---PLuJP---3.

FIG. 1. The repair of single strand breaks in helical DNA by polynucleotide ligase. In the reaction phosphodiester bonds, AMP
and 1’Pi are formed.

I’olynucleotide l&se, purified from extracts of Escherichia coli Given a DNA substrate containing single strand breaks’ such
infected with bacteriophage 1‘4 (l-3); catalyzes the covalent as those shown in Fig. 1, the enzyme catalyzes the repair of the
joining of two segments of an interrupted strand in a I>NA breaks by the formation of phosphodiester bonds; in the reaction
duplex, provided that no nucleotides are missing at the junction. ATP is cleaved to AMP and PPi.
Since the first description of T4 polynucleotide ligase (l), the
* This investigation was supported by Public Health Service
Research Career Program Award GM-13,634 and Research Grant purification procedure has been modified in order to obtain
AI-06045 from the National Institutes of Health, United States larger quantit,ies of the enzyme and to permit use of an ATP-PPi
Public Health Service.
$ Special Fellow of the United States Public Health Service, 1 DNA with single strand breaks refers to DNA treated with
Award GM-29,562. Present address, Department of Microbiology, pancreatic DNase in order to produce phosphodiester bond cleav-
The Johns Hopkins IJniversity Medical School, Baltimore, Mary- ages in the individual strands, but not to alter the molecular
land 21205. weight of the duplex DNA (4). Concatemers of X DNA refer to
0 Research Fellow of the International Agency for Research on linear aggregates of DNA prepared from DNA isolated from x
Cancer. bacteriophage as described in the preceding paper (4) ; r-+-ATP
7 Predoctoral Fellow of the National Science Foundation. refers to ATP labeled with 32P in the r-phosphoryl group.

4543
This is an Open Access article under the CC BY license.
4544 Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

32p ~ OH 32P OH allowed to cool slowly and then was dialyzed for 12 hours against
0.01 M Tris-HCI buffer (pH 8.0) containing 0.1 M NaCl. Be-
HO P32 HO- PS2
tween 60 and 70y0 of the molecules in these preparations were
circular, as measured by sucrose gradient sedimentation at
32P-PHOSPHOMONOESTERS, SENSITIVE
neutral pH (9).
TO PHOSPHATASE T-~~P-ATP was prepared as previously described (4). 2J4C-
dTTP, 3H-ATP, and 32P-AMP were purchased from Schwarz
BioResearch. Unlabeled nucleosides, nucleotides, and NAD+
were purchased from Calbiochem. dHMCTP was a gift from
Dr. A. Kornberg.
32P OH
Enzymes-Alkaline phosphatase (chromatographically puri-
HO PS2 fied) from E. coli (10) was purchased from Worthington and

0
further purified as described in the preceding paper (4). Phos-
32P -PHOSPHODIESTERS FORM ED, phodiesterase from the venom of Crotalus adarnanteus, spleen
RESISTANT TO PHOSPHATASE phosphodiesterase, and pancreatic DNase were products of
FIG. 2. Schematic representation of the standard assay for Worthington. Micrococcal nuclease (ll), purified from Micro-
polynucleotide ligase. coccus pyogenes, was a gift of Dr. C. A. Dekker. This prepara-
tion had a specific activity of 300,000 units per mg of protein when
exchange assay. The specific activity of the purified enzyme is assayed as previously described (12). Exonuclease I from E.
several-fold larger than previously described, and the enzyme co& a gift from Dr. I. R. Lehman, was the DEAE-cellulose frac-
preparation has been freed of contaminating deoxyribonuclease tion purified and assayed as described by Lehman (13). This
activity. This report describes the purification procedure in fraction had a specific activity of 9,000 unit,s per mg of protein.
detail and reveals several properties of the reaction which have The 5’-nucleotidase of C. adamanteus was purified and assayed as
not been previously reported. In these studies the modified described elsewhere (14). Crystalline X exonuclease (8) was a
DNA preparations and the techniques described in the preceding gift of Dr. A. D. Kaiser.
paper (4) have been used. A subsequent report (5) describes the Other Materials-3”Plabeled PPi was purchased from New
preparation and properties of an enzyme-adenylate complex as England Nuclear. DEAE-cellulose (Cellex D, exchange capacity
well as the evidence for its role as an intermediat’e in the ligase of 0.69 meq per g) was purchased from Bio-Rad. Whatman
reaction. phosphocellulose (exchange capacity of 3.7 meq per g) was
purchased from Reeve Angel Company (Los Angeles, California).
EXPERIMENTAL PROCEDURE Both resins were prepared for use by washing 100 g of powder
with distilled water by decanting and then equilibrating with
Materials
0.02 M Tris-HCl buffer (pH 7.4) (DEAE-cellulose) or 0.02 M
Nucleic Acids and NucZeotides-3H-Labeled and unlabeled T7 potassium phosphate buffer (pH 7.4) (phosphocellulose) .
DNA were prepared as described elsewhere (6). Unlabeled X Crystalline bovine plasma albumin was obtained from Armour.
DNA was prepared as previously described (4). 3H-X DNA was Dialysis tubing was boiled for 10 min in distilled water prior to
prepared as described by Gellert (7). T7 DNA and X DNA UW.
bearing 32P-5’-phosphoryl end groups and T7 DNA containing
Methods
external and internal 3Yphosphomonoesters (ligase substrate)
were prepared and characterized as described in Table VI of the Assay for Polynucleotide Ligase-This assay measures the
preceding paper (4). Salmon sperm DNA was a product of conversion of 5’-32P-phosphonlonoesters in DNA containing
Worthington. Where indicated, DNA4 samples were denatured single strand breaks into a form which remains acid-insoluble
by treatment with alkali as previously described (6). Unless after incubat,ion with phosphatase (Fig. 2). It is a modification
otherwise noted, concentrations of DNA are expressed as equiva- of the assay procedure described earlier (1). Unless otherwise
lents of nucleotide phosphorus. indicated, the reaction mixture (0.3 ml) contained 0.02 InM
Partially single stranded T7 DNA was prepared by limited 5’-32P-phosphoryl DNA2 (DNA Preparation I, Table VI (4)),
hydrolysis of native T7 DNA by incubation with X exonuclease 0.066 mM ATP, 66 mM Tris-HCl buffer (pH 7.6), 6.6 mM N’gCh,
(8). The reaction mixture (1.0 ml) contained 1.1 pmoles of T7 10 lllM dithiothreitol, and 5 X lop5 to 3 X lop4 unit of enzyme
DNA, 0.05 M glycine buffer (pH 9.2), 2 rant MgC&, and 100 diluted in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01 M
units of X exonuclease. Aliquots were removed from the reaction @-mercaptoethanol and 0.5 mg per ml of bovine plasma albumin.
mixture at intervals for measurement of the extent of hydrolysis After incubation for 20 min at 37”, 0.2 ml of salmon sperm DNA
by determining the amount of acid-soluble material with an (0.25 mg per ml), 0.5 ml of cold 0.6 M trichloracetic acid, and
absorption at 260 rnp. Incubation was continued until 29% of 2.0 ml of cold water were added in succession. After centrifuga-
the DNA was converted to acid-soluble material. The nuclease tion at 10,000 x g for 10 min, the supernatant fluid was discarded.
was removed by phenol extraction (4). Two milliliters of cold 0.01 M HCI were added to the tube, mixed,
Hydrogen-bonded concatemers of X DNA were prepared as and recentrifuged, and the supernatant fluid was discarded.
described in the preceding paper (4). Hydrogen-bonded The precipitate was dissolved in 0.5 ml of 0.1 M NaOH with the
circles of 3H-DNA were prepared by the procedure of Hershey, aid of a glass stirring rod. After incubation for 5 min at room
Burgi, and Ingraham (9). Tritium-labeled X DNA was diluted 2 The amount of DNA substrate added to the reaction mixture
to 4 pg per ml in 0.01 M Tris-HCI buffer (pH 8.0) containing 0.6 was adjusted to provide 0.6 rpmole of a’+‘-internal phosphomono-
M NaCl. After incubation at 75” for 2 min, the solution was esters.
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Sablon, T. R. Live, G. C. Fareed, and C. C. Richardson 4545

temperature, the solution was neutralized by adding 0.05 ml of 7.0

1.1 M HCl-0.2 M Tris. Alkaline phosphatase (10 pg) was added,
and each reaction mixture was incubat.ed for 30 min at 65”; t.hen 60
0.5 ml of cold 0.6 M trichloracetic acid and 2.0 ml of cold water
were added. The precipitate was collected on glass filters,
washed three times with 2-ml portions of 0.01 w HCl, and dried;
the radioactivity was determined as previously described (15).
The precipitate obtained from control incubations without
ligase contained 5% of the added radioactivity. The precipitate
obtained from control incubations with both ligase and phos-
phatase omitted contained 90% of the added radioactivity.
One unit of t’he enzyme is defined as the amount catalyzing
the conversion of 1 mpmole of 32P-phosphomonoesters into a
phosphatase-resistant form in 20 min. The activity was pro-
portional t,o enzyme concentration in the range given above.
Assay jar ATP-PPi Exchange Activity of Polynucleotide
Liguse-As discussed in the following paper (5), purified prepara-
tions of T4 polynucleotide ligase catalyze an exchange reaction
0 IO 20 30 40
between ATP and PPi. The standard assay measures the
conversion of 32PPi into a form which is adsorbable to Norit TIME AFTER INFECTION, MINUTES
The incubation mixture (0.3 ml) contained 3.3 pM 32PPi (specific FIG. Appearance of polynucleotide
3. ligase after infection of
activity 1 X IO5 cpm per pmole), 0.066 mM ATI’, 66 mM Tris- E. coli with phage T4. E. coli cells were grown in modified M-9
HCl buffer (pH 7.6), 6.6 mM MgC&, 10 mM dithiothreitol, and medium (see text). At a cell titer of 1 X lo9 per ml, L-tryptophan
0.01 to 0.1 unit’ of enzyme diluted as described above for t,he and five T4 r+ phages per cell were added (zero minutes). At
intervals, 40-ml -aliqyjots were pipetted onto ice, centrifuged, re-
ligase assay. After incubation at 37” for 20 min, the mixture was susDended in 0.05 M Tris-HCl buffer (aH 7.4)-0.001 M glutathione.
chilled to 0” and to it were added 2.0 ml of a solution containing and disrupted by sonic treatment. Ggase activity was measured
0.25 M HCl, 0.25 mM Pi, 0.25 mM PPi, and Norit (2y0 packed in the standard assay.
volume). The suspension was thoroughly mixed on a Vortex
DNA containing only phosphatase-resistant 32P was prepared
mixer and, after 5 min at O”, the Norit was collect,ed by filtration
by incubating the product of the ligase reaction described above
through a glass filter (Whatman GF/C glass paper, 2.4-cm
with alkaline phosphatase from E. coli. The incubation mixture
diameter). The Norit was washed with four 3-ml portions of
(2.0 ml) contained 0.3 pmole of DNA (ligase product’), 33 mM
0.01 M HCl. The papers were dried and the radioactivity was
Tris-HCl buffer (pH 8.0), and 0.1 mg of purified phosphatase.
determined in a thin window gas flow counter. One unit of
After incubation for 60 min at 65” the reaction was terminated
activity is defined as the amount catalyzing the conversion of 1
by chilling at 0”. The phosphatase was removed by phenol
mpmole of 32PPi into a Norit-adsorbable form in 20 min. The
extraction as previously described (6). Of the 32P initially
activity in Fractions V to VII was proportional to enzyme
present in the ligase product, 4Ooj, was removed by treatment
concent,ration in the range given above. The ratio of ATP-PPi
with phosphatase. Of the remaining 32P, 97% was found to be
exchange activity to ligase act,ivity in these fractions was 1.2 to
resistant to phosphat’ase after denaturation of the DNA.
1.6.
Other d(lethods-End group analysis of DNA, including the
Other Enzyme Jssays-a4ssays for cxonucleases I, IT, and III
measurement of external and internal phosphomonoesters in
and endonuclense I from E. coli were carried out as previously
helical DNA, was carried out as described in the preceding paper
described (16). The assay for E. coli DNA polymerase used
(4). Zone sedimentation of DNA in sucrose gradients was
l*C-dTTP as the labeled nucleoside triphosphate and poly dAT
carried out as previously described (6). Separation of mono-
as primer (17). The assay for the T4-induced DNA polymerase
nucleotides was accomplished by electrophoresis for 3 hours at a
was that previously described by Aposhian and Kornberg (18).
potential gradient of 20 volts per cm in 0.05 M sodium citrate
Assay for alkaline phosphatase was described in the accompany-
(pH 3.5). Separation of nucleoside mono-, di-, and triphos-
ing paper (4). Assay for 5’-nucleotidase was performed as
phates was accomplished by electrophoresis for 2 hours at a
previously described (14).
potential gradient of 20 volts per cm in 0.05 M sodium citrate
Preparation of DhrA Product of Ligase Reaction--,4 large scale
(pH 5.0). Whatman No. 3MM paper was used for electrophore-
preparation of the DNA product of the ligase reaction was carried
sis. Protein was determined by the procedure of Lowry et al.
out in order to obtain sufficient quantities for characterization.
The react,ion mixture (6 ml) contained 2.5 pmoles of 5’-32P- (19). Phosphate was determined by the method of Chen,
Toribara, and Warner (20).
phosphoryl DNA containing single strand breaks, 0.066 mM
ATP, 66 111~ Tris-HCl buffer (pH 7.6), 6.6 mM MgC&, 10 mM RESULTS
dithiothreitol, and 0.1 unit of polynucleotide ligase (Fraction VI).
Incubation was at, 37” for 45 min. The reaction was terminated Appearance of Ligase after Injection u;ith Phage T,$
by chilling to 0” and by adding 10 mM EDTA. The reaction Upon infection of E. coli with phage T4, there was at least a
mixture was then dialyzed for 3 days against five changes of 0.01 20-fold increase in ligase activity as measured in the standard
M Tris-HCl buffer (pH 7.6).1.0 1\1NaCl, and finally against 0.01 assay (Fig. 3). The T4-induced ligase appeared first about 5
M Tris-HCl buffer (pH 7.6)-0.05 M NaCl. Recovery OF radio- min after infection with a maximal level of activity (2.2 units
activity and of Dr\‘A was 95 yO. per mg of protein) observed approximately 20 min after infection.
4546 Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

TABLE I in place of ,&mercaptoethanol. The partially lysed cells were

Ligase activity in extracts Escherichia coli infected with different
o.f disrupted in 165-ml portions by sonic treatment for 10 min at a
bacteriophages maximum input in a Branson model 575 sonic oscillator. The
Extracts of E. coli B or E. coli CR63 infected with each of the extracts were centrifuged to remove cell debris and the super-
phages listed above were prepared and assayed for polynucleotide natant fluid (500 ml) was recovered. A sufficient volume (350
ligase as described under “Methods.” E. coli CR63 is the permis- ml) of the same buffer was added to the supernatant fluid to yield
sive host for T4 am H39X. a solution with an optical density of 68 at 260 rnp (Fraction I).
Host
Infecting Streptomycin Fractionation
bacteriophage
E. coli B E. coli CR63 To 850 ml of extract were added, with stirring, 170 ml of 5%
units/mg prottin
(w/v) strept.omycin sulfate over a 45-min period. After 15 min,
the suspension was centrifuged and the supernatant fluid was
None........................... <O.l <O.l
T4 recovered (Fraction II).
2.0 1.8
T2. 1.9 1.6 Ammonium Sulfate Precipitation
T4 am H39X (gene 30). . <O.l 0.9
Solid ammonium sulfate (320 g) was added gradually with
stirring to Fraction II (970 ml). The mixture was then stirred
TABLE II
for 30 min. The precipitate was collected by centrifugation and
Purijkation of Td-induced p olynucleotide ligase
dissolved in 100 ml of 0.01 M Tris-HCl buffer (pH 7.6)-0.1 M
step Ligase Protein Specific
Yield NaCl (FEaction III).
activity activity

lrnits units/mg DEAE-cellulose Fractionation

x IOF w/ml p7&i?& %
A column of DEAE-cellulose (25 cm2 x 20 cm) was prepared
I. Extract. 153 11.4 1.6 100
II. Streptomycin.. and washed with 9 liters of 0.01 M Tris-HCl buffer (pH 7.6).
98 5.0 2.0 64
III. Ammonium sulfate.. 48 15.2 2.8 Fraction III (1.7 g of protein) was dialyzed against 4 liters of
31
IV. DEAE-cellulose I., 38 3.5 3.6 25 this buffer for 12 hours, and was applied to the column. The
V. DEAE-cellulose II.. 16 0.15 14 IO adsorbent was washed with 1 liter of 0.01 M Tris-HCl buffer
VI. Phosphocellulose 9 <O.Ol 6 (pH 7.6) and the enzyme was eluted with the same buffer con-
VII. Concentration 8 0.08 1700 5 taining 0.3 M NaCl. The 0.3 M eluate was collected in loo-ml
fractions. Approximately 80% of the enzyme applied to the
A similar time course was observed when E. coli was infected adsorbent was obtained in 300 ml of the eluate (Fraction IV).
with T2 phage (Table I).
DEAE-cellulose Chromatography
In previous studies with conditional lethal mutants of T4, the
structural gene for polynucleotide ligase was found to be gene 30 A column of DEAE-cellulose (25 cm2 x 20 cm) was prepared
(21). As shown in Table I, T4 am H39X, a gene 30 mutant, and washed as before. Fraction IV (1 g of protein) was dialyzed
failed to induce an active ligase in E. coli B (the nonpermissive against 18 liters of the equilibrating buffer and applied to the
host) but did so in E. coli CR63 (the permissive host). These column. The column was washed with 1 liter of the same buffer.
results strongly indicate that the increased levels of ligase A linear gradient of elution (total volume of 9 liters) was applied
observed in E. coli infected with T4 are the result of a phage with 0.01 M Tris-HCl buffer (pH 7.6) and 0.01 M Tris-HCl buffer
enzyme. (pH 7.6)0.3 M NaCl as limiting concentrations. Fractions
(250 ml) were collected at 15-min intervals. Of the activity
Purification of T4 Polynucleotide Ligase applied to the column, 80% was eluted between 6.5 and 9.5 resin
Unless otherwise indicated, all procedures were carried out bed volumes of effluent (Fig. 4). The fractions containing
at O-5”, and all buffers contained 0.01 M P-mercaptoethanol. All enzyme of specific activity greater than 10 units per mg of
centrifugations were at 15,000 x g for 10 min. The results of protein were pooled to give Fraction V (750 ml).
a typical purification are given in Table II.
Phosphocelkulose Chromatography
Growth of Bacteria
A column of phosphocellulose (7 cm2 x 17 cm) was prepared
E. coli B was grown at 37” under forced aeration in a Fermacell
and washed first with 500 ml of 0.02 M potassium phosphate
(New Brunswick Scientific Company, Inc., New Brunswick, New
Jersey) in 50 liters of M-9 medium (22) containing casamino buffer (pH 7.6)0.6 M KCl, and then with 2 liters of 0.01 M
potassium phosphate buffer (pH 7.6)-0.1 M KCl. Fraction V
acids (2 g per liter). At a cell density of 2 x log per ml T4rf
(113 mg of protein) was diluted with an equal volume of 0.01 M
phages were added at a multiplicity of 4. Twenty minutes after
potassium phosphate buffer (pH 7.6)-0.10 M KC1 to yield a final
infection the culture was quickly cooled to 5”, and the cells were
harvested by centrifugation at 35,000 x g in a Sharples continu- volume of 1.5 liters. The diluted Fraction V was applied to the
column and the resin was washed with 220 ml of the same buffer.
ous flow centrifuge at a flow rate of 0.5 liter per min. The
gummy paste (105 g) was stored at -40” and showed no loss of A linear gradient of elution (total volume of 2 liters) was applied
with 0.01 M potassium phosphate buffer (pH 7.6)-0.10 M KCl,
activity after 9 months.
and 0.01 M potassium phosphate buffer (pH 7.6)0.50 M KC1 as
Preparation of Cell Extracts limiting concentrations. Fractions (22 ml) were collected at
Frozen cells (45 g, wet weight) were suspended in 480 ml of 4-min intervals. Of the activity applied to t,he column, 70 7Owas
0.05 M Tris-HCl buffer (pH 7.4) containing 0.001 M glutathione eluted between 9.5 and 12.5 resin bed volumes of effluent. The
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Xablon, T. R. Live, G. C. Fareed, and C. C. Richardson 4547

fractions containing more than 5% of the added enzyme activity

were pooled to give Fraction VI (132 ml).

Concentration of Enxyme
A column of phosphocellulose (1 cm2 x 5 cm) was prepared
and washed with 200 ml of 0.01 M potassium phosphate buffer
(pH 7.6)-0.10 M KCl. Fraction VI was diluted to a final volume
of 350 ml by the addition of 220 ml of 0.01 M potassium phosphate
buffer (pH 7.6). The diluted Fraction VI was applied to the
column and eluted with 0.01 M potassium phosphate buffer
(pH 7.6)0.5 M KCl. Of the activity applied to the column, 90%
FRACTION NUMBER
was eluted in a volume of 6 ml (Fraction VII).
The concentrated phosphocellulose fraction (Fraction VII) FIG. 4. Chromatography of polynucleotide ligase and ATP-
PPi exchange activities on DEAE-cellulose. The DEAE-cellulose
represented a lOOO-fold purification over the starting extract
I fraction (Fraction IV. 80 me of motein) was adsorbed and eluted
and contained 5% of the activity initially present. In all of the from phosphocellulose ‘as described in the text, except that the
experiments described this fraction was used as the enzyme resin bed volume of the column (column height kept constant) and
source unless otherwise stated. gradient volumes were one-tenth those described for the large
scale preparation. Fractions (36 fractions of 25 ml each) were
collected-and assayed for ligase.and ATP-PPi exchange activities
Assay of Polynucleotide Ligase during Purification as described under “Methods.” Of the ligase activity applied to
Accurate measurements of polynucleotide ligase in each frac- the column, 75y0 was recovered in Fractions 13 to 18.
tion of the purification procedure described above can be ob-
tained with the standard ligase assay. However, a more rapid activity during a storage period of 6 months at 0”. Fraction VII,
assay, which does not require the preparation of a special DNA made 50% with glycerol, was stored at -15” for 6 months with-
substrate, has been used to monitor the purification. As is out detectable loss in enzyme activity (less than 10 %).
discussed in a subsequent paper (5), purified preparations of
polynucleotide ligase catalyze an exchange reaction between ATP Absence of Other Enzyme Activities
and “PPi, the measurement of which provides a sensitive and The purified enzyme was free of deoxyribonuclease activity.
quantitative assay for the ligase activity (see “Methods”). When assayed under the optimal conditions for each nuclease,
Because extracts of E. coli contain other enzymes which there was no detectable exonuclease I (less than 5 units per mg
catalyze ATP-PPi exchange reactions, this assay cannot be used of protein), exonuclease II (less than 3 units per mg of protein),
until Step V when the partially purified enzyme is subjected to exonuclease HI (less than 15 units per mg of protein), or endo-
chromatography on DEAE-cellulose. As shown in Fig. 4, the nuclease I (less than 1 unit per mg of protein). For other studies
first peak of ATP-PPi exchange activity which elutes from the described in this paper it was important to determine whether
column coincides with the peak of ligase activity. Although not limited endonucleolytic action upon native DNA occurred which
shown in Fig. 4, a second peak of ATP-PPi exchange activity would yield fragments that were not acid-soluble. The intact-
which does not contain ligase activity elutes from the column at ness of the single strands of native DNA after exposure to 4 units
approximately 0.25 M NaCl. As previously described (23), the of the purified ligase under standard assay conditions was
only peak of ATP-PPi exchange activity eluted from the examined by observing sedimentation of the DNA (Fig. 5).
phosphocellulose column also coincided with the peak of ligase After incubation of native T7 DNA wit,h the enzyme, either in
activity. The ratio of ATP-PPi exchange activity to ligase the presence or absence of ATP, at least 90% of those strands
activity during the latter stages of purification is 1.2 to 1.6. which were intact before exposure to the enzyme remained so
Although the enzyme purification has been successfully after treatment.
repeated without the use of the standard ligase assay, it should be The concentrated phosphocellulose fraction had no detectable
emphasized that the frozen cells and reagents used in these 5’nucleotidase activity (less than 0.1% of its ligase activity)
purifications were the same. Therefore, any modification of the at pH 7.6. The purified enzyme contained no detectable
initial purification steps described in this paper should be first phosphatase activity; during incubation in the standard assay
monitored with t,he standard ligase assay. the enzyme (1.0 unit) released no acid-soluble radioactivity (less
than 5’%) from the DNA substrate containing 321’-labeled
Properties of Enzyme external and internal phosphomonoesters, either in the presence
or absence of ATP. There was no detectable hydrolysis of
Stability of Enzyme y-ATPa2; during a 50-min incubation with 1.0 unit of Fraction
Crude extracts of T4-infected cells could be kept at 0” for at VII in the absence of DNA substrate, less than 2y0 of the
least 1 week without significant loss in enzyme activity. All radioactivity was released into a form not adsorbed by Norit.
other enzyme fractions could be stored at 0” for 1 to 2 days DNA polymerase a,ssays showed no activity (less t’harl 1 unit
before proceeding to the next purification step without loss of per mg of protein) for either the T4 or E. coli DNA polymerases.
activity (less than 10 %). The phosphocellulose fraction
(Fraction VI), prior to concentration, has shown variable pH Optimum
instability. During storage at 0” Fraction VI has lost 50y0 of The optimal pH range for the purified enzyme is 7.2 to 7.8 in
its original activity in periods of from 2 to 6 weeks. The con- 66 mM Tris-HCl buffer. At pH 6.9 and 8.0 the enzyme showed
centrated purified enzyme (Fraction VII) lost 50 y0 of its original 46 and 65%, respectively, of its activity at pH 7.6.
4548 Enxymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

I I I I I Requirement for ATP

The enzyme has a specific requirement for ATP; in the absence
of ATP in the standard assay there is no detectable activity
(less than 5%). The requirement for ATP was confirmed by
the sedimentation analysis of T7 DNA containing single strand
breaks which was incubated with polynucleotide ligase and with
or without ATP; repair occurred only in the presence of ATP
(see Fig. 8 below). The K, for ATP, derived from plots ac-
cording to Lineweaver and Burk (24), is 1.4 X 1O-5 M. Of a
number of compounds tested none could replace ATP (Table III).
Except for dATP they did not inhibit the ligase when present at
a concentration of 0.066 mM in the standard assay containing
ATP. dATP, at a concentration of 0.066 mM, resulted in a 60%
decrease in activity. A plot of the reciprocal of the reaction
velocity against inhibitor concentration according to the method
of Dixon (25) gave a value of 3.5 x 10-5 M for the Ki and showed
it to be a competitive inhibitor of the enzyme. Of particular
interest is the inability of NAD+ to serve as cofactor since the
ligase purified from uninfected E. co&i has been shown to have a
specific requirement for this compound (26, 27).

Requirement for Single Strand Breaks in DNA

In order to assess the specificity of the enzyme, a number of
I 5 IO 15 20 DNAs were prepared containing different ratios of internal to
TUBE NUMBER external 32P-phosphomonoesters. The preparation and charac-
terization of these DNAs was described in the preceding paper
FIG. 5. Absence of endonuclease in purified polynucleotide (4). Each DNA was incubated with an excess of ligase, and the
ligase preparations as shown by zone sedimentation of DNA in percentage of the V-phosphomonoesters resistant to phospha-
alkali. Reaction mixtures (0.6 ml) contained 380 mpmoles of tase was determined. As shown in Fig. 6, with each DNA
native T7 DNA, 33 mM Tris-HCl buffer (pH 7.6), 6.6 mM MgCl;?, preparation the number of phosphomonoesters which became
6.6 mM dithiothreitol, and 0.066 mM ATP. Enzyme was omitted
from one reaction mixture (a) and 4 units of ligase (Fraction VII) resistant to phosphatase was nearly identical with the number
were added to reaction mixture (5). Incubation was for 30 min of internal phosphomonoesters initially present in the DNA.
at 37”. The reaction mixtures were dialyzed for 18 hours against For example, 2 and 90% of the 32P-phosphomonoesters in DNA
1 liter of 0.05 M NaCl-0.01 M Tris-HCl buffer (pH 7.6). The preparations containing 1 and 85%, respectively, of these groups
samples were then analyzed by zone sedimentation in alkali as
internally located were incorporated into phosphodiester bonds
described under “Methods.” Identical results were obtained
when ATP was omitted from the reaction mixture. in the ligase reaction. It has been previously shown that the
original external 52P-l,hosphomonoesters are not altered during
Eflect of Temperature on Enzymatic Rate
TABLE III
When the purified enzyme was assayed at O”, lo”, and 20”,
the rates were 10, 20, and 55%, respectively, of that
of reaction Requirement for ATP
observed at 37”. At 45“ only 20% of t.he activity seen at 37” Each compound (0.066 XLM) listed above was tested for its
was observed. However, the reaction was not linear with time, ability to serve as cofactor in the standard ligase assay. None of
the compounds, except dATP (see text), were inhibitory when
suggesting an inactivation of the enzyme at the elevated tem-
present at a concentration of 0.066 mM in the standard assay con-
perature. taining ATP.
Requirement for Divalent Cation Compound Activity

The purified enzyme requires added magnesium for activity.

mnpnwles/20 min/mg protein
In the absence of added MgClz there was no detectable activity
No addition. <50
(less than 5 y0 of maximal activity). Under the conditions of the ATP, 1500
standard assay the optimal Mg++ concentration was 10 mM. At GTP <50
3 and 30 m&f, 35 and SO%, respectively, of maximal activity were CTP <50
observed. Mn++ could only partially replace Mg++. Thus, at HTP. <50
an optimal Jlii++ concentration (10 mM), only 25% of the dATP. <lO
maximal activity seen with Mg++ was observed. dGTP. . <IO
dCTP. <lO
Sulfhydryl Requirement dTTP. <lO
dHMCT1’. <50
If dithiothreitol was omitted from the standard reaction
ADP. <50
mixture, only 257, of the maximal activity was obtained with
AMP <50
Fraction VII. When 10 mM /?-mercaptoethanol replaced dithio- NAD+ .. <50
threitol, 407, of the optimum activity was obtained.
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Sablon, T. R. Live, G. C. Fareed, and C. C. Richardson 4549

the ligase reaction (1). In an experiment in which 95 y0 of the TABLE IV

internal phosphomonoesters had been converted to a phospha- DNA substrate speciJicity of TQ ligase
tase-resistant form, all (more than 95%) of the original external T7 DNA with single strand breaks was the substrate routinely
32P-phosphomonoesters were still present as such. used in the standard ligase assay. All DNA preparations were
DNA preparations which do not contain internal phospho- treated with phosphatase and then labeled with a2P at their
monoesters, such as native T7 DNA and denatured T7 DNA, 5’-termini in the polynucleotide kinase reaction (see “Methods”).
could not serve as substrates for the ligase (Table IV). De- Where indicated, the DNA substrates were denatured as de-
naturation of the DNA containing internal phosphomonoesters scribed under “Methods.” Each DNA was tested for its ability
(substrate for the ligase) destroyed its ability to serve as a sub- to serve as substrate in the standard ligase assay at a concentra-
tion of 0.02 mM.
strate; less than 5% of its sZP could be converted to a phospha-
tase-resistant form even with an excess of enzyme. DNA substrate Activity

Rateand Extent of Phosphodiester Bond Formation mpmoles/Z0 ntin/mg protein

T7 DNA with single strand breaks. . 1700

Either with the addition of excess enzyme or with prolonged
Native T7 DNA. <IO
incubation, the formation of phosphodiester bonds reached an
Denatured T7 DNA. . <lO
apparent limit (Fig. 7). Further addition of enzyme resulted in T7 DNA with single strand breaks, dena-
no additional reaction. However, when an additional equal tured.................................. <lO
amount of DNA substrate was added to the reaction mixture, a
further reaction occurred equal in extent to that initially pro-
duced. The limit of the ‘-ieaction correlates closely with the l-
number of internal phosphomonoesters present in the substrate,
as discussed above.
The K, for DNA containing single strand breaks was found
t
be 1.5 x 1O-g pulexpressed as concentration of internal phospho- t
monoesters. The maximal velocity observed with saturating
concentrations of both ATP and DNA was 1.7 pmoles per 20
min per mg of protein.

‘O0i 1

I I I I I I
0 IO 20 30 40 50 60

TIME IN MINUTES
FIG. 7. Extent of phosphodiester bond formation during poly-
nucleotide ligase reaction. The incubations were carried out
under standard assay conditions in several tubes. Each tube
contained 5’-3eP-phosphoryl DNA (0.25 ppmole of 32P-labeled
internal phosphomonoesters) and 1 X 10e3 unit of polynucleotide
ligase. At 20 min an additional equal amount of DNA substrate
was added and the subsequent reaction was followed. Additional
enzyme (2 X lo+ unit) was added at 40 min of incubation.

Inability of Polynucleotide Ligase to Catalyze Joining of

Deoxyribonucleotides to DNA
J
20 40 60 80 100 If polynucleotide ligase could catalyze the joining of a deoxy-
% =P AT SINGLE-STRAND BREAKS nucleoside 5’-monophosphate to the 5’-phosphoryl terminus of
FIG. 6. Specificity of polynucleotide ligase for internal phospho- high molecular weight DNA, then, in the presence of all four
monoesters. Several preparations of 32P-labeled T7 DNA sub- deoxynucleoside 5’-mononucleotides and XTP, polymerization
strates containing varying ratios of labeled external phospho- of these monomers might occur at the ends of DNA. To provide
monoesters to internal phosphomonoesters were incubated with a possible DNA primer T7 DNA was partially degraded with X
an excess of polynucleotide ligase. The reaction was carried out
under standard assay conditions with replacement of the normal exonuclease to produce partially single stranded molecules. X
ligase substrate by 5 mpmoles (300 to 3000 cpm) of the DNAs to be exonuclease initiates hydrolysis from each 5’-phosphoryl end of
tested. The percentage of the total radioactivity that became a bihelical molecule, the progressive degradation leaving a
resistant to phosphatase is plotted against the percentage of the shortened helix with single stranded ends (28). If a mono-
total radioactivity which was originally present in internal
phosphomonoesters. The preparation and characterization of nucleotide becomes hydrogen-bonded to a complementary nu-
each labeled DNA substrate were the subjects of the preceding cleotide in the single stranded region, then polynucleotide ligase
paper (4). might catalyze the formation of a phosphodiester bond between
4550 Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

TARLE V lated that the incompleteness of the repair was due either to the
Decrease in internal ii’-termini in DNA during ligase reaction presence of double strand breaks in the DNA or to contaminat-
DNA containing single strand breaks was prepared by incu- ing deoxyribonuclease in the less purified ligase prepara-
bating T7 DNA with pancreatic DNase, and the numbers of ex- tion.
ternal and internal 5’-termini were determined as described in the As shown in Fig. 8, polynucleotide ligase can completely repair
preceding paper (4). Each DNA (300 mpmoles) was incubated T7 DNA containing single strand breaks. A sample of T7 DNA
in the standard ligase reaction mixture with 0.01 unit of poly- whose sedimentation rate in alkali had been reduced by incuba-
nucleotide ligase for 30 min. The reaction mixture was then tion with pancreatic DNase was incubated with polynucleotide
dialyzed against six changes of 1.0 M NaCl-0.01 M Tris-HCl buffer ligase and ATP. At least 90% of the fragmented DNA increased
(pH 7.6) and finally against 0.05 M NaCl-0.01 M Tris-HCl buffer
in sedimentation rate and acquired sedimentation properties
(pH 7.6). The numbers of internal and external termini were again
determined (4). The results are recorded as the number of indistinguishable from those of intact strands of T7 DNA (Fig.
termini per strand based on 40,000 nucleotides per strand of T7 8~). Furthermore, no DNA was found which sedimented at a
DNA (6). rate faster than that expected for intact single strands of T7
DNA. In a control experiment the helical DNA containing
External Internal
DXA 5.‘termini per strand 5’- termini per strand single strand breaks was incubated with the ligase in the absence
-
I I I I I
Experiment I
Substrate. 1.7 7.1 (a)
Product. 1.7 1.6
Experiment II 0.2
Substrate. 1.2 4.2
Product 1.1 <o. 1

the 3’-hydroxyl group of the nucleotide and the 5’-phosphoryl 2 0.1

terminus of the shortened strand of DNA. The presence of all
four deosgnucleotides in the reaction mixture would then permit c2
c-u
polymerization and the elongation of the chain in the 5’ direc-
tion. b
The reaction mixture contained 21 nipmoles of exonuclease- z 0.15
(b)
treated T7 DNA (see “Materials”) in a standard ligase reaction cn
mixture containing 20 mpmoles each of dGMP, dCMP, dTMP, 2 0.10
3H-dA?III’ (1 x lo7 cpm per mpmole), and 5 units of polynucleo- Ei
t,ide ligase. iYo detectable radioactivity (less than 1 pprnole of 0.05
dAMP) was converted into an acid-insoluble form in 60 mill of
2 0 E
incubation. Identical results were obtained during incubation
at 0”, lo”, or 37”, indicating that polynucleotide ligase cannot I-
catalyze the joining of mononucleotides to DKA. n
0 0.2
Characterization of DNA Product

Increase in Length of Polynucleotide Strands

End Qoroup Labeling-End group labeling of DNA with 0. I
polynucleotide kinase and y-32P-ATP provides a means for
molecular weight determination of linear polynucleotide strands
(29). This technique was used to show that the number of
internal 5’-end groups in a DNA preparation containing single 0 ‘-
strand breaks decreased during the ligase reaction. As shown in IO 15 20 25
Table V, in two experiments the number of internal 5’-termini in TUBE NUMBER
the DNA preparations decreased after incubation in the ligase
FIG. 8. Alkaline sedimentation analysis of the DNA product of
reaction. The number of external 5’.termini, on the other hand, the ligase reaction. T7 DNA was sedimented prior to any enzy-
remained unchanged. These results suggest that polynucleotide matic incubation (a), after treatment with pancreatic DNase
segments within a DNA duplex are covalently joined in the followed by incubation in the ligase reaction from which ATP was
reaction, whereas end to end joining of the duplex molecules does omitted (b), and after treatment with pancreatic DNase followed
not occur. by incubation in the complete ligase reaction (c). The T7 DNA
containing single strand breaks was the sample described in Fig. 4
Sedinzentation A~zalysis-Zone sedimentation provides an of the preceding paper (4). The DNA (500 mpmoles) was incu-
additional method for analyzing the DNA product of the ligase bated for 30 min in the ligase reaction (0.5 ml) with 0.2 unit of
reaction. Earlier it was shown by sedimentation analysis that Fraction VII. A control incubation contained 0.2 unit of ligase
T7 DNA containing single strand breaks could be partially re- but no ATP. After dialysis for 12 hours at 4” against 0.05 M NaCl-
0.01 M Tris-HCl buffer (pH 8.0)) the DNA was denatured and 100 to
paired by incubation with polynucleotide ligase and ATP (1). In 300 mpmoles were analyzed by zone sedimentation analysis as
those studies only 50y0 of the individual DNA strands acquired described under “Methods.” All DNA preparations were de-
sedimentation properties of the intact strands. It was postu- natured prior to sedimentation.
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Xablon, T. R. Live, G. C. Fareed, and C. C. Richardson 4551

of ATP; no detectable change in its sedimentation properties

(Fig. 8b) was observed.
Previous experiments had shown that external 32P-phospho-
monoesters were not altered in the ligase reaction, suggesting

I
that end to end joining of duplex molecules did not occur. This
finding was confirmed by sedimentation analysis. The sedi- LIGASE
mentation behavior of the duplex DNA, either native or con- SYSTEM
taining single strand breaks (same as in Fig. 8), at neutral pH,
was unchanged by incubation in the ligase reaction (Fig. 9).
XYZABC
Formation of Phosphodiester Bonds
Hydrolysis of Product to S’- and 5’-Nucleoside Monophosphates-
A product of the ligase reaction, containing only phosphatase-
resistant 32P (see “Methods”), was degraded with nucleases of SPLEEN VENOM
known specificity in order to determine the nature of the linkage
DIESTERASE DIESTERASE
formed during the ligase reaction. A schematic representation
/ \

Z A

S-MONONUCLEOTI DES 5’-MONONUCLEOTI DES

FIG. 10. Scheme for nearest neighbor analysis of phosphodiesters
formed in the ligase reaction.

TABLE VI
Nearest neighbor analysis of nucleotides joined in ligase reaction
A T7 DNA preparation (300 mrmoles) containing 90% of its
Y? in internal phosphomonoesters was incubated in the standard
ligase reaction mixture for 30 min with 0.01 unit of polynucleotide
ligase. An aliquot of the reaction mixture was subjected to the
standard assay procedure; 90% of the s2P in the DNA became
insusceptible to phosphatase. The incubation mixture was
dialyzed as described in Table V and then incubated with 0.05 mg
of phosphatase for 30 min at 65” to remove any remainiug phos-
phomonoesters. The protein was extracted with phenol and the
DNA was dialyzed against four changes of 0.01 M Tris-HCl buffer
(pH 7.6)-0.05 M NaCl. Samples of both the DNA substrate and
product were hydrolyzed to their constituent nucleoside 5’-
monophosphates by the consecutive act.ion of pancreatic DNase
and snake venom phosphodiesterase (4) or to nucleoside 3’.mono-
phosphates by the action of micrococcal and spleen nucleases
(see Fig. 10). Identification of the labeled nucleotides was per-
formed as previously described (4). Recovery of radioactivity in
each experiment, relative to the unhydrolyzed DNA, was greater
than 85%.
(d) __..- ._._......
DNA product DNA
Sucleotide substrate
(3’.nucleotides)
3’.Nucleotides 5’.Nucleotides

% % %
A
dAMP 31 22 22
dTMP 44 59 57
5 IO 15 20 11 8 9
dGMP
TUBE NUMBER dCMP 14 11 12

FIG. 9. Sedimentation of duplex DNA products of ligase reac-

tion. Native T7 DNA and T7 DNA containing single strand
breaks were sedimented prior to incubation in the ligase reaction of the results is shown in Fig. 10. When the DNA product was
(a and c, respectively) and after incubation in the ligase reaction hydrolyzed with pancreatic DNase and snake venom phos-
(6 and d, respectively). The DNA preparations, conditions of phodiesterase, more than 95y0 of the radioactivity was recovered
incubation, and dialysis are described in Fig. 8. The DNAs were
as nucleoside monophosphate (Table VI). On the basis of the
not denatured and sedimentation analysis was performed at
neutral pH as described under “Methods.” From 150 to 200 specificity of these enzymes, one expects to find the 5’-isomers
mpmoles of DNA were present in each gradient. of the nucleoside monophosphates (30, 31). This was confirmed
 Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

TABLE VII phosphates as described previously (Fig. 10). As shown in Table

Analysis of exonuclease I digest of ligase reaction encl product VI, the 5’-nucleotides which were released were identical with
Samples of the DNA substrate and product described in Table those present as internal 5’-terminal residues prior to joining.
VI were incubated in separate reaction mixtures containing exo- The distribution of radioactivity in the nucleoside 5’.monophos-
nuclease I from Escherichia coli. The reaction mixtures (0.3 ml) phates is clearly not random; approximately SOY0 of the label
contained 150 mMmoles of DNA substrate or product, 0.05 M was present in dTMP. Since the ligase reaction went to com-
glycine buffer (pH 9.2), 0.01 M MgC12, and 300 units of exonuclease pletion, this distribution must reflect specificity of pancreatic
I. After incubation for 1 hour at 37”, measurement of trichlor- DNase (4) and possibly that of polynucleotide kinase. A
acetic acid-soluble material on aliquots of each mixture revealed relatively high amount of dTMP (44%) was also found in the
t,hat more than 9570 of the radioactivity had been rendered acid-
soluble. 5’-Nucleotidase (5 units) was then added to each reac- 3’-residues that participated in the ligase reaction. While the
tion mixture, and incubation was continued for 30 min. Each proportion of dTMP was smaller than that in the 5’-residues, the
reaction mixture was then assayed for the formation of Norit- proport,ion of dAMP was correspondingly higher.
nonadsorbable radioactivity (a2Pi) as previously described (12). It is of interest to note that of the four nucleotides present as
This value was taken as the amount of 5’-mononucleotides formed either 3’- or 5’-residues, approximately 80% are dAMP and
by exonuclease I treatment. The amount of acid-soluble radio- dTMP. These results may reflect a specificity of the pancreatic
activity that was not susceptible to 5’-nncleotidase is recorded as DNase and polynucleotide kinase for these particular nucleotides
dinucleotides. or perhaps a preference of the enzymes for AT-rich regions in
DNA Dinucleotides Mononucleotides helical DNA.
Hydrolysis of Product by E. coli Exonuclease I-The location of
70 totalradioaclivily the a2P was confirmed by analysis of the products following
Substrate. >95 <5 hydrolysis of the DNA product by exonuclease I from B. coli (13).
Product. 9 91 This enzyme is unable to hydrolyze the phosphodiester bond of a
I
dinucleotide (13). Thus, exonuclease I will init.iate a stepwise
TABLE VIII hydrolysis from the 3’-hydroxyl terminus of a single stranded
DNA molecule, liberating 5’-mononucleotides, but will leave
lnco,poration of 5’-32P-phosphomonoesters in concatemers of X
DhrA into phosphodiester bond linkages the 5’-terminal dinucleotides of the molecule intact. This
property of exonuclease I has previously been used to establish
Samples of X DNA bearing 5’-S2P-phosphomonoesters were
prepared and characterized as described in Table VII of the the terminal location of the 32P incorporated into DNA during
preceding paper (4). Samples of each DNA preparation (10 to the polgnucleotide kinase reaction (15). A similar experiment
30 mpmoles of DNA-P) were incubated in standard ligase reaction showed that more than 95oj, of the radioactivity in a ligase
mixture containing 0.2 unit of ligase. The measurement, of 3%’ substrate (DNA containing internal 5’-32P-phosphomonoesters)
incorporated into phosphodiester bonds was performed as in the was present as dinucleotides after hydrolysis by exonuclease I
standard assay. (Table VII). However, after incubation in the ligase reaction,
Internal 32P. Phosphodiester more than 90% of the radioactivity was present in mononucleo-
X DXA preparation phosphomonoesters bonds formed tides following digestion by exonuclease I (Table VII). This
-
% % finding shows that the 32P-phosphomonoesters at internal ter-
mini in t,he substrate were incorporated into nonterminal phos-
Concatemers . . 35 25
Linear monomers. . 8 5 phodiesters which were then hydrolyzed by exonuclease I to
Denatured. .._._ .._._ ._. <5 <5 yield 5’-mononucleotides.
Formation of Covalently Joined Concatemers and
by incubating the labeled nucleotides released by the above Circles of Phage X DNA
treatment with the specific 5’-nucleotidase from C. adamanteus In the preceding paper (4) it was shown that external 5’-32P-
venom; 95 y. of the radioactivity was released as Pi. phosphomonoesters of X DNA could be converted to internal
When the DNA sample was hydrolyzed with micrococcal and phosphomonoesters by procedures favoring the formation of
spleen nucleases, more than 95% of the radioactivity was again concatemers. If the 32P-phosphomonoester of any molecule in
released as nucleoside monophosphates (Table VI). In this such a concatemer is in juxtaposition to the 3’-hydroxyl end
instance, however, the nucleoside monophosphates should be group of the next, then they should be incorporated into phos-
the 3’-isomers (32, 33). As expected, the nucleotides were not phodiester linkages by polynucleotide ligase. When a prepara-
susceptible to the action of the 5’-nucleotidase but were hy- tion of X DNA concatemers bearing 5’-32P-phosphomonoesters
drolyzed by the relatively nonspecific alkaline phosphatase from was incubated in the ligase reaction, 75% of the internal phos-
E. coli (more than 90% hydrolysis). The results, considered phomonoesters were incorporated into phosphodiester bonds
together, show that 3’,5’-phosphodiester bonds are formed at (Table VIII). In control experiments with preparations con-
the sites of joining by polynucleotide ligase. t.aining predominantly linear monomers or denatured DNA, the
Nearest Neighbor Analysis of Nucleotides Joined in Ligase 32P incorporated into phosphodiester bonds was considerably
Reaction-During the ligase reaction internal 32P-labeled 5’- lower (Table VIII).
phosphomonoesters in the DNA substrate become linked The purified ligase also catalyzes the conversion of hydrogen-
to the 3’-carbons of the adjacent nucleotides. As a result, it bonded circles of X DNA to covalently closed molecules, a form
is possible to identify the nucleotides which are joined by the which can be readily identified by a 4-fold increase in sedimenta-
new phosphodiester bonds (34). This was accomplished by tion rate in alkali (35). When a sample of 3H-X DNA was con-
digesting the DNA to yield either 3’- or 5’-nucleoside mono- verted to hydrogen-bonded circles as described under “Materials,”
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Sablon, T. R. Live, G. C. Fareed, and C. C. Richardson 4553

sedimentation analysis at neutral pH revealed that 50 y0 was now more accurate estimation of the number of phosphodiester bonds
in this form. After incubation of the hydrogen-bonded circles formed during these reactions.
in a ligase reaction, 20% of the total DNA was converted to a
form whose sedimentation rate in alkali was 4-fold greater than Attempts to Reverse Ligase Reaction
that of hydrogen-bonded X circles (Fig. lla). This result sug- Several experiments have been carried out to determine the
gests that 40% of the original hydrogen-bonded circles were reversibility of the polynucleotide ligase reaction. In the fol-
converted to the covalently closed form. Furthermore, in lowing paper (5) it is shown that an enzyme-adenylate complex
addition to the formation of covalently closed circles, sedimelita- serves as an intermediate in the ligase reaction. Therefore, one
tion analysis in alkali (Fig. lib) also revealed the formation of step in the reaction may involve the formation of a phospho-
single stranded cyclic monomers (15%). In control experiments diester bond and the release of AMP. A reversal of the ligase
in which ligase was omitted from the reaction mixture, all of reaction would result in the hydrolysis of a phosphodiester bond
the DNA (greater than 95%) had sedimentation characteristics in t.he presence of enzyme and AMP.
in alkali of linear monomers. A sensitive assay for detecting phosphodiester bond cleavage
The relatively low yield of covalently closed molecules ob- is to look for the production of single strand breaks in native T7
served in this experiment is in part due to the presence of single DNA as measured by sedimentation analysis of the denatured
strand breaks in the DNA preparation. Thus sedimentation DNA. Native T7 DNA was incubated in the standard ligase
analysis of the X DNA in alkali revealed that from 20 to 33% of reaction containing 1 unit of ligase and 0.2 rnM AMP instead of
the DNA strands contained one interruption or more (Fig. 1%). ilTP. Examination of the DNA after denaturution by zone
It is also possible that some of the DNA molecules have been sedimentation analysis did not reveal any detectable fragmenta-
subjected to a limited hydrolysis at their termini by phospha- tion of the DNA (less than 10% increase in the amount of slower
tases or exonucleases; either modification may render t,he hy- sedimenting material). Ident.ical results were obtained when
drogea-bonded circles ineffective as substrates for the ligase 0.01 mM PPi was also present in the reaction mixture together
reaction. wit,h AMP.
The ability of the T4 polynucleotide ligase to catalyze the
formation of covalently closed circular molecules from hydrogen- DISCUSSION
bonded circles has been previously shown by Becker et al. (2).
Similar findings have also been obtained for the ligase purified Enzymes which catalyze the covalent joining of single strand
from uninfected E. coli (7, 36-38). These studies showed that interruptions in bihelical DNA have been identified and purified
at least 65y, of the hydrogen-bonded circles of X DNrl can be from uninfected B. coli, T4-, and T7-infected B. coli (l-3, 26, 36,
converted into a covalently closed form. Experiments with X 38). The identification and purification of these DNA ligases
DNA bearing 5’-??-phosphomonoesters (Table VIII) provide a have been facilitated by the development of a number of different
assays with such substrates as hydrogen-bonded circles and
concatemers of X DNA molecules (7, 26, 36), 5’-32P-l~hosphoryl
DNA (1, 2, 36, 38), polynucleot,ides covalently linked to cellulose
(3), and transforming DNiz (39). 9’ L ~iice several of ll:c iissays
are t,ime-consuming, and since the DNA substrates may not be
readily available in all laboratories, a procedure not requiring
the measurement of joining activity has been developed for the
purification of the T4 polynucleotide ligase. The reproducibility
of the early purification steps obviates enzyme assays in all but
the chromatographic procedures. During these latter purifica-
tion steps a rapid and simple assay has been used which measures
the ability of the ligase to catalyze an exchange between PPi and
ATP (5).
The ligases purified from uninfcctcd E. coli and from E. coli
infected with T4 phages have many ljroperties in common
FRACTION NUMBER
(l-3, 26, 36, 38). 130th polynucleotide ligases are specific for
FIG. 11. Alkaline sedimentation analysis of covalently joined the same DNA substrates and produce the same DNA end
circles of x DNA. Covalent h circles were prepared by incubating product,s. The reactions catalyzed by both enzymes are medi-
hydrogen-bonded circles of X DNA with polynucleotide ligase. ated by an enzyme-adcnylate complex (2, 5, 23, 40), although the
The reaction mixture (0.37 ml) contained 0.9 mpmoles of hydrogen-
bonded circles of 3H-x DNA (2.6 X lo7 cpm per pmole), 30 mM Tris- adenylate moiety is derived from different cofactors. As
HCl buffer (pH 7.6), 5 mM MgG, 5 mM 2.mercaptoethanol, 0.05 described in this paper, the cofactor for the T4-induced enzyme
mM ATP, and 1 unit of ligase. After incubation for 1 min at 25” is ATP, while that for the enzyme isolated from uninfected E.
the reaction mixture was chilled to 0”. The incubation mixture coli has been shown to be NAD+ (26, 27). Furthermore, the
was made 12 rnM with Na$DTA and then 0.1 M with NaOH. I<, values for the respective cofactors for the two enzymes are
Equal volumes (0.22 ml) of the solution were layered on 5-ml alka-
line sucrose gradients (5 to 20% sucrose) containing 0.7 M xacl- strikingly different. Thus, Olivera and Lehman (27) have re-
0.3 M n’aOH-0.01 M Tris-HCl-0.001 M EDTA. Tube a was cen- ported a K, value of 1 X lop7 M for NAD+ for t’hc ligase from
trifuged for 75 min and tube b for 225 min at 38,000 rpm in the uninfected cells, while Zimmerman et al. (26) reported a value of
Spinco SW 39 rotor. Fractions (3 drops) were collected from the 3 x lo-* RI for t,he same cofactor. However, the K, for ATP in
bottom of the tubes and O.I-ml aliquots were pipetted onto glass
filter paper, dried, and counted in a liquid scintillation counter. the reaction catalyzed by the T4 ligase was found to be 1.4
Recovery of radioactivity was greater than 85%. X lop5 M, as reported in this paper. ny contrast, the affinity
 Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI Vol. 243, No. 17

for the DNA substrates appeared t,o be very similar for the two The nature of the reaction catalyzed by 1’4 polynucleotide
enzymes. ligase suggests several possible roles for the enzyme in viva.
Like many other enzymes of nucleic acid metabolism which First, the enzyme could be involved in the process of genetic
have been purified and characterized, the T4 ligase provides an recombination. Studies on bacteriophages have shown that
enzymatic reagent for studying nucleic acid struct,ure and metab- genetic recombination involves the enzymatic breaking and
olism. For example, the ability of a given DNA to serve as a rejoining of preformed polynucleotide strands (45, 46). After
substrate for the enzyme indicates that the DNA contains single infection of E. coli by phage T4, parental phage DNA undergoes
strand breaks displaying 5’-phosphoryl and Y’-hydroxyl end fragmentation and dispersion among other parental and progeny
groups not separated by missing nucleotides. In confirmat’ion molecules. Anraku and Tomizawa (47) and Kozinski, Kozinski,
of the work of others (2, 26, 36-38), the results described in this and James (48) have isolated recombinant molecules of T4 DNA
paper with T4 ligase indicate that these structural features are joined only by hydrogen bonds and have shown that the single
present in hydrogen-bonded X circles and concatemers. Sim- strand interruptions in these molecules are eventually repaired.
ilarly, recent studies have shown that the specifically located Although polynucleotide ligase may be involved in the final
single strand int,erruptions in native T5 DNA (41) are quan- covalent joining of the polynucleotide fragments through phos-
titat.ively repaired in the polynucleotide ligase reaction (42). phodiester bond linkage, studies with ligase-negative mutants
Information of this type concerning the structure of DNA mole- of phage T4 have thus far failed to reveal any defect in the forma-
cules would be difficult to obtain by any other method now tion of recombinant molecules.3 Similarly, the presence of T4
available. ligase could explain in part the presence of concatemers (49) of
The 3’- and 5’-terminal nucleotides at the sites of single T4 DNA observed in ,%‘.coli cells infected with T4 phage. Like-
strand breaks in these interrupted DNAs can be identified if end wise, physical and genetic studies on t,he repair of ultraviolet-
group labeling is used in conjunction with polynucleotide ligase, irradiated DNA (50) have suggested a terminal step in which
followed by nearest neighbor analysis of the product. This phosphodiester bond formation occurs.
approach can be applied not only to the st.udy of naturally occur- Finally, it is possible that polynucleotide ligase functions in
ring phosphodiester bond interruptions such as those found in DNA replication. Genetic and autoradiographic studies indicate
hydrogen-bonded circles of X DNA or in T5 DNA, but it may a linear, sequential replication of the bacterial chromosome
also be used to study interruptions produced by nucleases. For starting from one point on the molecule (50). Studies in z&o,
example, in the present study the specificity of pancreatic DNase however, have indicated that purified preparations of E. coli
was examined under conditions in which only a few phospho- DNA polymerase fail to copy both strands of a duplex molecule
diester bonds were cleaved in a duplex molecule. The technique simultaneously from one end of the helix and, as a consequence,
is of particular value in the characterization of nucleases that there are aberrations in the structure of the DNA product (51).
produce one or a very few specific interruptions in DNA mole- One model proposes that the replication of DNA in viva can
cules. Finally, the ability t,o introduce 32P at a specific site in a occur without assuming the existence of an additional polymerase
naturally occurring DNA provides a unique substrate for the or assigning novel catalytic properties to the purified polymerase
detection of enrymcs which cleave the molecule at this specific (52). Since the genetic and autoradiographic analyses men-
site. tioned above do not permit resolution at the nucleotide level, it
Polynucleotide ligase has also provided information on the is possible that one or both strands of the chromosome are
ability of DNA polymerase to repair single stranded regions in a synthesized in a discontinuous manner. According to this model,
circular duplex. Gcfter, Becker, and Hurwitz (36) were able to synthesis of both strands of chromosomal DNA occurs in a 5’ to 3’
show that nucleotides deleted from hydrogen-bonded circles of direction, but only short segments of DNA are synthesized at a
X DNA by cxonuclcase III could be replaced by E. coli DNA time. These relatively short polynucleotides would then be
polymerase, thus restoring the DNA to a substrate for poly- covalently joined in a reaction similar to that catalyzed by
nucleotide ligase. Likewise, Goulian and Kornberg (43) used polynucleotide ligase. Experiments by Okazaki et al. (53) with
E. coli DNA polgmerase to replicate single stranded circular growing fi. coli B and with T4-infected E. coli B suggest that
DNA isolated from phage A\113 or +X174; treatment of the small molecular DNA subunits with sedimentation coefficients
product lvith polynucleotide ligasc produced a fully covalent of 9 to 11 S are intermediates in the formation of long chains of
duplex circle. DNA, thus lending some support to t,he above model of DNA
The studies of Olivera and Lehman (38) and Cozzarelli et al. (3) synthesis.
with purified ligases indicate further that homopolymers are At present the precise function of polynucleotide ligase in viva
free to slide along complementary homopolymers until t.he 5’. is not known. However, studies with conditional lethal mutants
phosphoryl terminus of one chain assumes a position adjacent to of phage T4 defective in the synthesis of polynucleotide ligase
the 3’.hydrosyl terminus of another. Gupta et al. (44) have (21) have provided some information about the biological role of
found that polynucleotidc ligase is able to catalyze the joining of this enzyme.3 E. coli 13 infected with these mutant’s synthesizes
thymidine oligonucleotides containing 7 thymidylate residues or a limited amount of phage-specific DNA under nonpermissive
more provided that polydeoxyadenylate of high molecular weight conditions (54, 55). The newly synthesized DNA is of low
is present. These investigators have also succeeded in joining molecular weight as measured by sedimentation analysis at
two deosyribooligonucleotides of known but different sequence alkaline pH, but can be converted to a form with a faster sedi-
in the presence of the appropriate complementary deoxyribo- mentation coefficient by incubation with purified ligase prior to
nucleotides. These experiments make it clear that t’he organic denaturation.3 In addition, this DNA appears to be the pre-
synthesis of small oligonucleotides followed by enzymatic joining cursor of mature T4 DNA. These results suggest that the T4
provides a promising aljproach to the synthesis of high molecular a Y. Masamune and C. C. Richardson, manuscript in prepara-
weight polynuclcotides of defined sequence. tion.
Issue of September 10, 1968 B. Weiss, A. Jacquemin-Sal&n, T. R. Live, G. C. Fareed, and C. C. Richardson 4555

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