PHOSPHOROLYSIS OF OLIGORXBONUCLEOTIDES
  BY POLYNUCLEOTIDE PHOSPHORYLASE

               BY MAXINE F. SINGER*
(From the National Institute of Arthritis and Metabolic Diseases, National Institutes of
    Health, United States Public Health Service , Bethesda, Maryland)

        (Received for publication, December 23 , 1957)

The enzyme polynucleotide phosphorylase catalyzes the over-all reaction

described in Equation 1.

n nucleoside-PP . Mg* (nucleoside-P), + n inorganic P (1)

This reversible reaction has been demonstrated With enzyme preparations
from Azotobacter trinelundii (1, 2) and Escherichia coli (3).  In the forward
direction the enzyme catalyzes the formation of polyribonucleotides sim-
ilar in structural details to natural RNA1 (5-8).  In the reverse reaction
polyribonucleotides are phosphorolyzed to yield nucleoside diphosphates.
The phosphorolysis of synthetic polymers made by the enzyme and the
phosphorolysis of ribonucleic acids isolated from various natural sources
have been studied by Ochoa and coworkers with the A. VineZundii enzyme
(2, 9), by Littauer and Kornberg with a preparation from E. coli (3), and
by Heppel.2  Highly polymerized RNA preparations were phosphorolyzed
at slower rates than the synthetic polymers; however, commercial RNA
and RNA "core," the limit polynucleotides obtained after exhaustive

* Research Fellow of the National Institute of Arthritis and Metabolic Diseases,
United States Public Health Service.
1 The following abbreviations are used: adenosine and uridine units are repre-
sented by A and U, respectively; 5`-diphosphates of adenosine and uridine, ADP
and UDP ; 5'-nucleoside diphosphate, NDP; 5'-monophosphates of adenosine and
uridine, AMP and UMP; 5`-triphosphate of adenosine, ATP; ribonucleic acid, RNA;
polyadenylic acid, poly A; polyuridylic acid, poly U; mixed polymer of adenylic and
uridylic acids, poly AU; tris(hydroxymethyl)aminomethane, Tris; reduced diphos-
phopyridine nucleotide, DPNH; inorganic orthophosphate, Pi.  Small polynucleo-
tides are designated by a system proposed by Markham and Smith (4).  A phosphate
group is designated by "p"; when placed to the right of a nucleoside symbol, the
phosphate is esterified at C-3' of the ribose moiety; when placed to the left of the
nucleoside symbol, the phosphate is esterified at (2-5` of the ribose moiety.  Thus,
pApA is a dinucleotide with 1 phosphate monoesterified at C-5` of an adenosine
residue and a phospho diester bond between C-3' of that same adenosine residue and
(2-5' of the other adenosine group.  The symbol -cyclic-p is used to designate a
terminal 2',3'-cyclic phospho diester moiety on a polynucleotide.  Thus, UpU-
cyclic-p is a dinucleotide with a 2',3'-phosphate at the terminal ribose.

2 Cited by Littauer and Kornberg (3) and Ochoa (9).

211

212 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

digestion of RNA with pancreatic ribonuclease, were attacked very slowly,
if at all.  In the light of these data it was not possible to define any require-
ments, by polynucleotide phosphorylase, for specific structural details in
the polynucleotide substrates.  The preparations used were not of uni-
form, known chain length and, in addition, the end group structures of the
natural RNA preparations have not been clearly established.

Heppel and coworkers recently described methods for the preparation of
several homologous series of well characterized oligoribonucleotides (7, 8,
10).  This made it possible to investigate the specificity of the phosphoro-
lysis reaction.  The experiments to be described in this paper indicate that
polynucleotides with C-5' phospho monoester end groups are readily phos-
phorolyzed although those bearing (3-3' phospho monoester end groups are
resistant to enzymic attack.  The phospho monoester at (2-5' is not es-
sential for activity, however, since oligonucleotides with no monoesterified
phosphate groups, such as trinucleoside diphosphates, are phosphorolyaed.
In addition, it was found that dinucleotides and dinucleoside monophos-
phates are not attacked by polynucleotide phosphorylase, and these com-
pounds accumulate as resistant end products when the phosphorolysis of
the larger oligonucleotides is studied.

A preliminary report of these findings has been made (11).

EXPERIMENTAL

Materials-Polynucleotide phosphorylase was purified from E. coli by
Dr. R. J. Hilmoe according to the procedure of Littauer and Kornberg (3).
The fraction used is described as Ethanol I (3) and was dialyzed before use.
The preparation contained 1 mg. of protein per ml. and had a specific ac-
tivity of 15, determined by the "exchange" assay (Assay C) (3).  The
preparation of A. vinelandii polynucleotide phosphorylase was kindly sup-
plied by Dr. s. Ochoa.  The fraction was an eluate from Ca3(P04)z gel
(12), contained 7.4 mg. of protein per ml., and had a specific activity of 40
as measured with the "exchange" assay (Assay 2) (2).  Crystalline bovine
pancreatic ribonuclease was a commercial preparation (Armour, lot No.
1044). Phosphomonoesterase was fractionated from human seminal
plasma (4) after a preliminary treatment with protamine to remove nucleic
acid material.  The specific activity of this preparation was 3.7 X lo3
units per mg. of protein (1 unit of enzyme liberates 1 pmole of inorganic
phosphate per hour per 1.2 ml. of a reaction mixture containing 26 pmoles of
AMP, in acetate buffer, pH 5.2).  This enzyme preparation dephosphoryl-
ated dinucleotides more slowly than mononucleotides.  Therefore, for the
preparation of dinucleoside monophosphates a great excess of enzyme was
used.  Even more enzyme was required to dephosphorylate oligonucleo-
tides of chain length greater than 2.  The preparation used in these experi-

M. F. SINGER 213

ments was relatively free from phosphodiesterase activity, but, when it
was used in the high concentrations required to dephosphorylate oligonu-
cleotides, appreciable diesterase activity was noted.  The nuclease from
guinea pig liver nuclei, which liberates small polynucleotides with C-5'
phosphate end groups from poly A, has been described by Heppel and co-
workers (10) .a  A commercial preparation of lactic dehydrogenase (Worth-
ington, crystalline) which was contaminated with pyruvate kinase was used
as a source of both of these enzymes.  Myokinase (63 units per ml., 0.8
mg. of protein per ml.) was supplied by Dr. B. L. Horecker and was pre-
pared according to the procedure of Colowick (13).

Polymers were prepared from nucleoside diphosphates with the poly-
nucleotide phosphorylase of E. coli or A. winelandii according to the pro-
cedure of Grunberg-Manago and coworkers (2), but the method of isolation
was slightly modified.  After polymer formation at pH 8, the reaction mix-
tures were adjusted to pH 7 with acetic acid.  Polymer was precipitated
with 3 volumes of ethanol, separated by centrifugation, dissolved in water,
and reprecipitated in the same manner.  The twice precipitated polymer
was dissolved in water and the solution was deproteinized by shaking with
0.25 volume of chloroform and 0.1 volume of isoamyl alcohol (14).  The
aqueous solution of polymer was then dialyzed against cold, running, dis-
tilled water for 3 days and lyophilized.  A sample of poly A prepared with
the polynucleotide phosphorylase of Micrococcus Zysodeikticus (15) was
generously donated by Dr. R. F. Beers.

All the mononucleotides used were commercial preparations (Sigma).
Phosphoenolpyruvic acid was prepared by Mr. William E. Pricer, Jr., and
DPNH was donated by Dr. B. L. Horecker.
Paper Chromatography and Paper Electrophog"esis-Severa1 systems were
used for the separation of mononucleotides and oligonucleotides on paper.
Descending chromatography was carried out with the following solvent
systems: System 1, isopropanol-water (70: 30, v/v) with NHI in the vapor
phase (16) ; System 2, isobutyric acid-1 M NHIOH-0.2 M ethylenediamine-
tetraacetate (100: 60:0.8, v/v/v) (17).  For Solvent 1, Whatman No. 3MM
paper was used and for Solvent 2, Whatman No. 3MM or Whatman No. 1.
Electrophoretic separations (referred to as System 3) were carried out ac-
cording to Markham and Smith (16) on strips (57 X 10 cm.) of Whatman
No. 3MM paper saturated with 0.05 M ammonium formate buffer, pH
3.5.  A potential of lo00 volts was applied across the paper.  Purine- and
pyrimidine-containing compounds were located on the paper strips with an
ultraviolet light which was also used to photograph the strips.  When
these methods of separation were used in a preparative way or to obtain
materials for enzymic treatment, the purine and pyrimidine derivatives

*We thank Dr. R. J. Hilmoe and Dr. L. A. Heppel for this preparation.

214 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

were eluted with water.  When quantitative elution of the material was
desired, the ultraviolet-containing areas were eluted with 0.01 N HCl for
6 hours at room temperature, and the concentration of the compound was
determined by measuring the absorption of the eluate at an appropriate
wave length.  From the adjacent region of the paper strip, an area of identi-
cal size was cut out and eluted in order to correct for the ultraviolet-ab-
sorbing material present in the paper itself.

Assay Procedures-In the procedure used (3), the phosphorolysis of
polynucleotides was carried out in the presence of inorganic P32.  The
resultant labeled nucleoside diphosphate (see Equation 1) was separated
from inorganic Pa2 by adsorption onto charcoal and its radioactivity was
measured.  The reaction mixtures (0.125 ml.) contained the oligonucleo-
tide or polymer, enzyme, 5 pmoles of Tris buffer, pH 8.0, 0.5 pmole of
MgC12, and 3.2 pmoles of P32-labeled sodium potassium phosphate buffer,
pH 7.4.  After incubation at 37", the reaction was stopped by adding 0.1
ml. aliquots to 1.0 ml. of cold, 2.5 per cent perchloric acid.  Acid-washed
Norit A (0.1 ml. of a 10 per cent suspension, w/w) was added to adsorb the
nucleotides.  After 10 minutes in the cold, the suspension was centrifuged
and the charcoal was washed three times with 2.5 ml. portions of water.
The charcoal was then suspended in 0.8 ml. of 50 per cent ethanol contain-
ing 0.3 ml. of concentrated NH,OH per 100 ml.  An aliquot of this sus-
pension (usually 0.1 ml.) was placed on a copper planchet, dried, and the
radioactivity determined with a thin window, gas flow counter.  The total
counts per minute incorporated into charcoal-adsorbable material were de-
termined and from the specific radioactivity of the inorganic Pa2 the micro-
moles of phosphate incorporated were calculated.  A self-absorption factor
of 1.15 (3) was applied.  Two control incubations, one containing no sub-
strate and one containing no enzyme, were generally carried through the
whole procedure with each experiment, and the results presented have been
corrected for the small amount of radioactivity adsorbed onto the charcoal
from these samples.

When the products of phosphorolysis were to be isolated, the reactions
were carried out in the same manner but on a larger scale.  Nucleotides
were eluted from charcoal by four treatments with 0.8 ml. of ethanolic
ammonia; the ethanol supernatant fluids were pooled and concentrated
before chromatography.

The phosphorolysis of polynucleotides was also measured by determining
the nucleoside diphosphate formed with the spectrophotometric procedure
of Kornberg and Pricer (18).  The application of this method to the de-
termination of polynucleotide phosphorylase activity has been described
by Ochoa and Heppel (6).  Myokinase was added to the assay system to
reconvert to ADP any AMP and ATP that had been formed in the original

M. F. SINGER 215

incubation as a result of myokinase activity in the polynucleotide phos-
phorylase preparation.  UDP was also measured by this method (19).
All optical measurements were carried out with a Beckman model DU
spectrophotometer .

Preparation of Oligonucleotides-Five series of homologous oligonucleo-
tides were prepared in order to study the specificity of polynucleotide phos-
phorylase.  Each series included compounds of 2, 3, and 4 nucleoside
residues and the structure of a representative member of each group is
illustrated in Fig. 1.  In every compound studied, the internucleoside links
were 3f, 5f-phospho diester bonds.  The oligonucleotides used in this work
were recently investigated by Heppel and coworkers (7,8, lo), who isolated
them from biosynthetic polymers and characterized them by methods
developed by Markham and Smith (4), Volkin and Cohn (20), and others.

The compounds in Group I contained adenosine as the only nucleoside
moiety and each of the three oligonucleotides had a monoesterified phos-
phate at C-5' of the terminal nucleoside (7, lo).  The structure of the
trinucleotide, pApApA, is shown in Fig. 1.  These compounds were de-
scribed and characterized by Heppel and coworkers (7, 10) and are formed
by the action of a nuclease from guinea pig liver nuclei on synthetic poly
A.  A typical preparation was carried out in the following way: The incuba-
tion mixture (30 ml.) contained 400 pmoles of MgC12, 800 pmoles of phos-
phate buffer, pH 7.2, 80 mg. of poly A (M. Zysodeikticus), and 5 ml. of
enzyme containing about 30 mg. of protein.  After 6 hours of incubation
at 37", toluene was added to inhibit bacterial growth.  After 24 hours of
incubation the mixture was cooled, deproteinized with chloroform (14), and
concentrated by lyophilization.  The resulting solution was divided into
eight equal portions and each was applied as a thin band to filter paper
and chromatographed for 64 hours in System 1.  The di-, tri-, and tetra-
nucleotides (pApA, pApApA, and pApApApA) were eluted from the papers
and the eluates were concentrated at 40" in a stream of air.  The concentra-
tion of oligonucleotide was estimated by measuring the ultraviolet absorp-
tion of the eluate at 257 mp and applying the extinction coefficient for
adenylic acid.4  The dinucleotide (pApA) that was obtained in this way
was contaminated with ADP and inorganic P and was further purified by
paper electrophoresis.

  It was assumed for this work that the molar extinction coefficient of an oligo-
nucleotide is approximated by the sum of the molar extinction coefficients of its
constituent nucleotides.  For example, the concentration C of ApApUp in micro-
moles of oligonucleotide per ml., when the absorption is measured at pH 2, is given
by C = Ab~.~~~/(2(15.1) + 10.0), where the molar extinction coefficients for AMP
and UMP are 15,100 and 10,000, respectively.  Recent unpublished experiments by
the present author indicate that, after alkaline hydrolysis, the absorption of pApApA
at 257 mp and pH 7 increases by approximately 15 per cent.  Enzymic hydroIysis of
dinucleotides in the deoxyribose series also results in an increased absorption (21).

216 PHOSPHOROLYSIS OF OLIGONUCLEOTZDES

The compounds of Group I1 were derived from those of Group I by the
removal of the monoesterified phosphate at (3-5' by human seminal plasma
phosphomonoesterase (7, 10). The structure of the trinucleoside diphos-

T*e

C

I

C

M2f"l""

F@w

Trinucleotide of Qvup I

4w

Trinucleoside diphosphate of Oroup 11

P

P.

   UpVpO-cyc=C-P
Trinucleatids of mnlp v

FIG. 1. Outline structures of the trinucIeotides and trinucIeoside diphosphates

phate (ApApA), which was derived from pApApA, is shown in Fig. 1.
The di-, tri-, or tetranucleotide of Group I was incubated with monoesterase
under the conditions described by Heppel and coworkers (7), and the cor-

M. F. SINGER 217

responding nucleoside derivatives were separated by chromatography in
System 1 (10).  The products were obtained from the paper strips in the
manner described for the compounds of Group I.

The three oligonucleotides in Group I11 were obtained by exhaustive
digestion of poly AU with ribonuclease (8).  The structure of the trinucleo-
tide, ApApUp, is shown in Fig. 1.  These oligonucleotides contained vary-
ing numbers of adenosine residues but each had a terminal uridine unit
with a monoesterified phosphate at its C-3'.  Thus the homologous di-
and tetranucleotides were ApUp and ApApApUp, respectively. The
electrophoretic and chromatographic properties of these compounds and
the methods of identification have been recorded by Markham and Smith
(4) and Volkin and Cohn (20).  In a typical preparation, 15 mg. of poly
AU were incubated with 0.6 mg. of ribonuclease for 16 hours at 37" in
0.05 M Tris buffer, pH 8 (total volume equal to 1.7 ml.).  The mixture was
deproteinized as described above (14), and the aqueous layer was concen-
trated, applied to paper, and chromatographed in System 1.  The bands
obtained were eluted and the absorption of the eluates at 260 mp was de-
termined.  The sum of the extinction coefficients for each mononucleotide
residue was used to estimate concentration of the oligonucleotide?

The dinucleoside monophosphate ApU, trinucleoside diphosphate
ApApU, and tetranucleoside triphosphate ApApApU of Group IV were
obtained from the corresponding oligonucleotides of Group 111 by treat-
ment with seminal phosphomonoesterase, exactly as described for the
preparation of Group 11 from Group I.  The properties of the compounds
in Group IV are described in the literature (8, 4, 20) and differ from those
of Group I1 in that the terminal nucleoside is uridine (see Fig. 1).  The
concentrations of these compounds were estimated as described for Group
111.

The last homologous series of oligonucleotides (Group V) was obtained
from the controlled digestion of poly U with small amounts of ribonuclease
(7).  Each member of the group contained exclusively uridine residues
with a cyclic-terminal phospho diester moiety.  The structure of the cyclic
terminal trinucleotide, UpUpU-cyclic-p, is shown in Fig. 1.  These com-
pounds were separated by chromatography in Solvent 1 and were eluted
from the papers with water.  By applying the extinction coefficient for
uridylic acid,' the concentrations of the oligonucleotides were determined
by measuring the absorption of the eluates at 262 mp.

Results

The trinucleotide and tetranucleotide of Group I, which had a phospho
monoester group at C-5', were readily phosphorolyzed by the polynucleo-
tide phosphorylases of E. coli and A. Vinektndii (Table I).  The compounds

218 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

pole per ml
  0.3
  0.3
  0.4

0.4
0.4
0.3

0.9

of Group 111, which contained a phospho monoester moiety at C-3', were,
however, resistant to phosphorolysis by either of the enzymes (Table 11).
The data in Table I1 suggested that ApApApUp was very slowly cleaved
by the A. vinehdii enzyme, but this was not confirmed by other studies
with both enzyme fractions.  Incubation ww carried out as in Table II
but for a period of 24 hours; chromatographic investigation of the reaction

           TABLE I
Phosphorolysis of Compounds in Group I and II

I I

E. coli experiment

Substrate 1 Substrate I

  1 Rateof
concen- radi- phospho-
tration. actlvlty+ rolysist

Group No.

~

I

I1

1 Poly A

c.9.m.
  0
3470
8910

0
1899
6080

4620

0.0
0.6
1.4

0.0
0.3
1 .o

9.3

A. &clundii experiment

Substrate
concen-
tration*

mole pa ml.
0.6
0.6
0.7

0.7
0.7
0.9

0.9

'otal radi.
mctivityt

c.p.m.

130
3,700
12,780

  18
2,280
6,380

3,880

Rate of
hosphor-
olysist

0.0
0.7
2.6

0.0
0.5
1.3

7.4

* Concentrations are expressed in terms of adenosine equivalents.  To obtain the

t Total number of counts per minute adsorbed onto charcoal.

$ Micromoles of phosphate incorporated into charcoal-adsorbable compounds per
hour per mg. of protein.  In the experiment with the E. coli enzyme, each tube con-
tained 786,000 c.p.m. as Pial and 26 y of protein except for the poly A incubation
mixture, which contained 2 y of protein; incubation time, 1 hour.  In the experiment
with the A. vinelandii enzyme, each tube contained 556,000 c.p.m. as Pi32 and 14 y
of protein except for the poly Aincubation mixture, which contained 1.5 y of protein;
incubation time, 2 hours.  The other components of the reaction mixtures and the
procedures are described under the section on methods.

concentration as oligonucleotide, divide by the chain length.

mixtures failed to reveal any significant breakdown of ApApApUp.  The
remaining data in Tables I and I1 show that, although the C-3' phospho
monoester moiety inhibits the phosphorolysis of an oligonucleotide, the
C-5' phospho monoester group is not a structural requirement for enzymic
action.  Thus ApApA and ApApApA (Table I) as well as ApApU and
ApApApU (Table 11) were phosphorolyxed at rates that do not differ
greatly from those found with pApApA and pApApApA.  It was also
found that the three compounds in Group V, which contain the terminal
cyclic-p, were completely resistant to enzymic attack.

M. F. SINGER 219

Only qualitative conclusions may be drawn from the relative rates of
phosphorolysis given in Tables I and 11.  InsufEcient amounts of material
made it impossible to conduct the experiments at saturating substrate
concentrations and only the following limited conclusions concerning rates
can be drawn : (1) with equivalent concentrations of active oligonucleotide,
the tetranucleoside derivative is phosphorolyzed more rapidly than is the

            TABLE I1
Phosphorolysis of Compounds in Group 111 and IV

Group No.

I Ir

IV

Substrate

Poly A

E. coli experiment

Substrate
concen-
tration*

mots )er ml.
0.4
0.3
0.3

0.3
0.3
0.3

0.9

*ob! radi.
lac tivi tyt

c.9.m.

0
18
110

0
644
2600

1781

Rate of
phospho-
rolyaist

0.0
0.0
0.0

0.0
0.2
0.8

10.4

A. vinelandii experiment

Substrate
concen-
tration.

mole 9.w ml.
0.7
0.8
0.8

0.6
0.6
0.3

0.9

'otal radi
lactivityt

c.P.m.
399
340
1682

115
4640
3145

3365

* Concentrations are expressed in terms of nucleoside equivalents.  1
the concentration, as oligonucleotide, divide by the chain length.
t Total number of counts per minute adsorbed onto charcoal.

Rate of
phospho-
rolysist

0.0
0.0
0.1

0.0
0.4
0.3

5.6

obtain

$ Micromoles of phosphate incorporated into charcoal-adsorbable compounds per
hour per mg. of protein.  In the experiment with the E. coli enzyme, each tube con-
tained 544,000 c.p.m. as Pisa and 20 y of protein except for the poly A incubation
mixture, which contained 1 y of protein; incubation time, 1 hour.  In the experiment
with the A. winelandii enzyme, each tube contained 628,000 c.p.m. as Pia2 and 30 y
of protein except for the poly A incubation mixture, which contained 1.5 y of pro-
tein; incubation time, 2 hours.  The other components of the reaction mixtures and
the procedures are described under the section on methods.

trinucleoside derivative, (2) the rates of phosphorolysis of corresponding
members of the several groups are of the same order of magnitude, as for
example pApApA, ApApA, and ApApU.  Data on the phosphorolysis of
poly A are included as a standard for comparison of rates and were obtained
at a concentration of polymer which afforded a maximal reaction rate.
Preliminary experiments carried out with pApApA indicated that a con-
centration of 2.3 pmoles per ml. as adenosine units (0.8 pmole per ml. as
trinucleotide) was sufficient to saturate the E. coli enzyme.  When both
were tested at saturating concentrations, the rate of phosphorolysis of
pApApA exceeded that found for poly A by a factor of approximately

220 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

2-fold.  The phosphorolysis of oligonucleotide attained a maximal rate at

The production of nucleoside diphosphates as one of the products of the
phosphorolysis of oligonucleotides was demonstrated by the spectrophoto-
metric assay as well as by the chromatographic evidence discussed below.

M inorganic P and required Mg++ ion.

              TABLE I11
Production of Nucleoside Diphosphates from Oligonucleotides

Experiment
  No.

Substrate

Substrate
concentration*

lynolw per ml.
  1.8
  1.6

1.4
1.3
0.6

0.5
0.6
0.5

Pia incorporated
into charcoal
adsorbable

I,rmok
0.00
0.13

0.14
0.03
0.04

0.00
0.02
0.04

Nucleoside
diphosphate
produced

pm Is
0.00
0.14

0.19
0.03
0.04

0.00
0.02
0.04

* Concentrations are expressed in terms of nucleoside units.  To obtain the con-
centration, as oligonucleotide, divide by the chain length,  The incubation mixtures
were as described in the section on methods, except that the scale was doubled (final
volume, 0.25 ml.).  The E. coli enzyme was added in the following amounts: 20 y in
Experiments 1 and 2, and 30 y in Experiment 3,  After incubation for 2 hours at 37O,
0.1 ml. was treated as described for the standard assay to determine the micromoles
of Pi** incorporated into charcoal-adsorbable compounds.  Another 0.1 ml. aliquot
was diluted to 0.5 ml. (Experiment 1) or 0.4 ml. (Experiments 2 and 3) with HtO,
heated for 2 minutes at 100", centrifuged to remove the protein, and 0.30 ml. aliquots
of the supernatant fluid were used to determine ADP or UDP.  The components of
the assay system (1.00 ml. in a cuvette with a 1.0 cm. light path) were 5 pmoles of
MgC12, 0.4 pmole of phosphoenolpyruvate, 0.1 pmole of DPNH, 0.005 ml. of myo-
kinase, and 0.005 ml. of lactic dehydrogenase containing pyruvate kinase.

The data in Table 111 show that the amount of inorganic Ps2 incorporated
into charcoal-adsorbable compounds was equal to the nucleoside diphos-
phate formed.
It was found that dinucleotides and dinucleoside monophosphates did
not undergo phosphorolysis (Tables I, 11, and 111).  This was true regard-
less of the nature of the end group on the compound containing 2 nucleoside
units.  A similar conclusion was reached when the phosphorolysis of
pApApA and pApApApA was studied as a function of time and the reac-
tions were allowed to go to completion.  Fig. 2 shows that poly A was

M. F. SINGER 221

I I
0       0

A polymer -

-

-

-

I

e 4

PAPAPAPA -

PAPAPA -

-

W

e

-

I 1

phosphorolyzed approximately 100 per cent; that is, the inorganic PS2 in-
corporated into charcoal-adsorbable nucleotides when the reaction stopped
was equivalent to the micromoles of polymer (expressed as adenine res-
idues) originally added.  With the trinucleotide, however, inorganic Pa
uptake ceased when 1 pmole of phosphate had been consumed per 3 pmoles
of adenine residues, or, to express this in another way, when 1 phosphate
molecule had been incorporated per molecule of trinucleotide (Fig. 2).

This suggested that the reaction proceeded according to the following
equation
           PAPAPA + Pi + PAPA + ADP            (2)

and came to a halt when the limit dinucleotide wa.9 formed.  Similarly,
with the tetranucleotide, the incorporation of inorganic Paz ceased when 2
pmoles of phosphate were taken up per micromole of tetranucleotide (Fig.
Z), again indicating that the dinucleotide is an end product of phosphoroly-
sis.  It should be mentioned that the accumulation of such a limit di-
nucleotide during the phosphorolysis of poly A would have been too small
to detect in this experiment.

A dinucleotide tentatively identified as cyclic dianhydrodiadenylic acid
was also tested as a substrate for E. coli polynucleotide phosphorylase.

222 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

Neither the chemically prepared material (22) nor that obtained from mam-
malian tissues (23) was phosphorolyzed by the
Identification of Reaction Products-Chromatographic investigation of the
reaction products confirmed the identification of the nucleoside diphos-
phates and also showed that a dinucleotide or dinucleoside monophosphate,
depending on the starting substance, accumulates during the phosphoro-
lysis of trinucleoside or tetranucleoside derivatives.  Reaction mixtures
were treated as described under the section on methods and nucleotide
material was eluted from the washed charcoal by successive treatments
with ethanolic ammonia.  The eluates were chromatographed in appropri-
ate solvents, along with suitable markers, and the ultraviolet-absorbing
spots were then eluted and treated in various ways to confirm their identity.
In one typical experiment, the substrate was pApApApA and the charcoal
eluate was chromatographed in solvent System 2. Fig. 3, a is a photograph
of that chromatogram.  The markers are on the left and the reaction mix-
ture on the right.  The most rapidly moving area (c) contained the di-
nucleotide pApA and will be discussed in detail.  The next area (b) cor-
responds in Rp to ADP and the slowest moving area (a) to ATP.  ATP
arises from ADP owing to myokinase present in the E. coli enzyme prep-
aration, and the AMP that is also formed is mixed with pApA in the fastest
moving area.  The ADP area (b) was eluted and its specific radioactivity
determined.  The data in Table IV give the specific activity of the ADP
as well as that of the ADP and UDP isolated by similar techniques from
the phosphorolysis of the other oligonucleotides.  In each case, the spe-
cific radioactivity of the nucleoside diphosphate was equal to that of the
inorganic Paz present in the incubation mixture.  Included in Table IV are
the Rp values of authentic samples of the nucleoside diphosphates and those
found for the isolated materials.  As indicated in Table IV, this procedure
did not afford quantitative recovery of the products.

In the experiment with pApApApA (Fig. 3, a) the material with the
highest Rp in Solvent 2 (Area c) was eluted and portions of it were subjected
to analysis by Systems 1 and 3.  A photograph which demonstrates the
paper electrophoresis of this material is shown in Fig. 3, b.  The known
markers are indicated; the eluate from Area c of Fig. 3, a had the mobility
of pApA.  In System 1 the major portion of the material also behaved as
pApA (Table V).  Similar techniques were used to identify pApA as a
product of the phosphorolysis of pApApA, and ApA as a product of the
phosphorolysis of ApApA, ApApApA, ApApU, and ApApApU (Table V).
The solvent system used for the initial separation of the dinucleotide or

6 We are indebted to Dr. Markham for a sample of the synthetic material and to
Dr. Rall for a sample of the isolated compound.

Jd. F. SINGER 223

dinucleoside monophosphate from the nucleoside diphosphate is shown in
Table V; the material was eluted from the paper and then subjected to the
other treatments indicaked.  The dinucleotides and dinucleoside mono-

PIG, 3. (a), uitraviolet photograph of paper chromatogram showing the products
of phosphoroiysis of pApAphpA.  The reaction mixture (0.15 mi. voIame) contained
5pinoles of Tris buffer, pfi 8,O.Spmole of MgC12,3.2 &moles of 1'132 containing 596,oOO
c.p,ni., 20 y of E'. coli enzyme, and 0,3 rmole of pApApAp.4 (expressed us adcnosiric
tinits).  Incubation time, 1.5 hours at 37".  The entire mixture was treated us de-
scribed in the section on methods and the washed charcoal was eluted three times
with 0.8 ml. of ethanolic ammonia.  The eluate was concentrated, applied to paper,
and chromstogruphed for 20 hours in System 2.  The known compounds are on tahe
left.  (b) , ultraviolet photograph of paper electrophoresis strip demonstritting pApA
3s ii product of llie phosphorolysis of pApApA4pA.  Area c of the chrornittogrurn
shwn in (a) was eluted with water, concentri&ed, and aul-tjected to p:tper electro-
phoresis (System 3) for 2 hours.

phosphates isolntsed were devoid of any radioactivity.  The phpA iso.
li~ted from the phosphorolysis of pApApA was, in addition, treated with
semen monoesterme as described for the preparation of the compounds of
Group 11.  The product hpA was identified by cmmatogmphy in Solvent
1, where it had an Rsp-aMp (R, relative to the Rp of 5'-AMP) equal to
2.23.  The Rs~-~~~~ of known ApA mas 2.23, and that for pApA was 0.42.

                    TABLE IV
Identification of ADP and UDP as Produets of Oligonucleotide Phosphorolysis

pApApA.. . . .. .
pApApApA.. . . .
ApApA ... . . . .. .
ApApApA.. . . .
ApApU ... . . . . . .
ApApApU. .. ...

Substrate

Concen-
trationt

0.3
0.3
0.6
0.5
0.7
0.6

RF of
JDP pro-
duced'

0.42
0.36
0.48
0.30
0.17
0.22
0.45

Rr'-AMP
authentic
compound$

0.40
0.44
1.9
2.0
2.0
2.0

NDP
produced

z;tff

0.45
0.43
1.9
2.0
2.2
2.2

Rp of
uthentii
NDP*

0.37
0.36
0.43
0.29
0.19
0.22
0.46

Mobility

compound

NDP
eluted

pmolc

0.024
0.014
0.041
0.064
0.110
0.108
0.045

Mobility
found

NDP

ADP

(6

64

I(

UDP

ADP

I<

c.9.m. peY

pwlc
150,OOO
210,000
130,000
150,000
170, OOO
160,OoO
140,000

6.p.m.

3,790
2,m
5,400
9,700
18,400
16,800
6,500

180,000
190,000
140,OOO
150,OOO
160,000
160,000
160,OOO

* Rp values in solvent System 2.
t Specific radioactivity, in counts per minute per micromole, of inorganic P3*
present in the incubation mixture.

$ Substrate concentrations are expressed as nucleoside units present in a 0.15 ml.
incubation mixture.  These experiments were carried out in a manner similar to
that described for the experiment of Fig. 3.  The E. coli enzyme was used.  The
data for pApApApA here were obtained from that experiment.  The nucleoside
diphosphate area on the chromatogram was quantitatively eluted, its ultraviolet
absorption determined to give the micromoles of ADP produced, and the radioactiv-
ity of the eluate was determined on a suitable aliquot.  Similar manipulations gave
the data for the other compounds.

                    TABLE V
Identification of Dinucleotides and Dinucleoside Monophosphates as Products
         of Oligonucleotide Phosphorolysis

System 2'

System 1

System 3t

Limit
dinucleoside
derivative

Substrate

RP
authentic
compound

RF
found

0.48
0.47
0.73
0.62
0.70
0.74

0.48
0.49
0.73
0.67
0.74
.O .74

9.1
10.9
4.7

8.9
10.9
4.6

4.0 1 4.4

* System used to separate dinucleoside derivative from nucleoside diphosphate

t Mobility is calculated as cm. per 2 hours per lo00 volts applied.

$ R6um gives the Rp relative to the Rp of 5'-AMP. The dinucleoside derivative
end products were obtained from the same ehperiments described for Table IV, or
from similar experiments.  The experiment for pApApApA is described in detail in
the text and in Fig. 3.  The charcoal eluates were chromatographed in System 2 to
separate the products, the pApA and ApA areas were eluted and concentrated, and
samples were subjected to chromatography in System f and paper electrophoresis,

224

and nucleoside triphosphate.

M. F. SINGER 225

In one experiment, all the products of phosphorolysis of pApApA were
eluted quantitatively from a paper chromatogram run in System 2, and the
yield of each substance was determined.  It was assumed, for the purpose
of calculation, that the pApA was contaminated by an amount of AMP
equivalent to the ATP found, since both would have been formed in equal
quantities from ADP by the action of myokinase.  It was then possible
to determine that the ratio of adenylic acid units recovered as pApA to
those recovered as the sum of ATP, ADP, and AMP was 2.2.  The theo-
retical value given by Equation 2 is 2.0.

DISCUSSION

The data presented here describe the specificity of polynucleotide phos-
phorylase with respect to the nature of the end groups on a polynucleotide
substrate.  It is clear that oligonucleotides which contain a monoesteri-
fied phosphate at C-3' of the terminal nucleoside residue (ApApUp and
ApApApUp) are resistant to phosphorolysis, and the same is true for the
three oligonucleotides bearing a terminal 2`, 3'-cyclic phosphate.  By con-
trast, phosphorolysis occurs readily with comparable compounds which
possess phosphate monoesterified at C-5` (pApApA and pApApApA) or
contain no phospho monoester groups at all (for example, ApApA, ApApU).

The resistance of relatively degraded commercial yeast RNA and RNA *
"core" to phosphorolysis (3) is consistent with the present results, for
these preparations are known to contain C-3' end groups (20, 24).  Other
factors such as molecular size must be considered, however, when compar-
ing the rates of phosphorolysis of different RKA and biosynthetic polymer
preparations.  Ochocz has demonstrated that the state of aggregation of
the polymer chains influences the rate of phosphorolysis (9).  In the pres-
ent work, comparisons have been made between oligonucleotides of the
same chain length, so that the influence of end group structure could be
studied without the complication of gross differences in molecular weight.

A study of the products of phosphorolysis of oligonucleotides yields
additional information concerning the mechanism of the polynucleotide
phosphorylase reaction.  The cleavage of the trinucleoside diphosphate,
ApApA, can be used as an example.  Phosphorolysis can occur in one of
the two ways illustrated in Equations 3 and 4:

ApApA + Pi 4 ApA + ADP

ApApA + Pi --.) adenosine + ppApA

(3)

(4)

Equation 3 involves cleavage at a nucleoside unit linked to the chain by
its C-5'-hydroxyl, and the products are the pyrophosphorylated mono-
nucleotide (ADP) and ApA.  In Equation 4 phosphorolysis occurs at the

226 PHOSPHOROLYSIS OF OLIGONUCLEOTIDES

nucleoside linked by its C3'-hydroxyl to the rest of the polynucleotide, and
the products are adenosine and the pyrophosphorylated oligonucleotide,
ppApA.  In the experiments described above, the products of the phos-

phorolysis of ApApA were ApA and ADP, indicating that the reaction
proceeded according to Equation 3. Similarly, the phosphorolysis of
ApApApU produced UDP and ADP, not adenosine and ADP, and the
cleavage of the other oligonucleotides also proceeded according to Equation
3.  This mechanism represents the reverse of a polymerization mechanism
in which the 5'-nucleoside diphosphate units are added to the C3'-hy-
droxyls of the acceptor nucleotides with the displacement of inorganic P
from the mononucleotide.  This phosphorolysis, according to Equation 3,
is equivalent to the reverse of the polymerization Mechanism B discussed
by Kornberg (25).  Polymerization Mechanism A (25) represents the re-
verse of Equation 4, and does not appear to be applicable to this enzyme,

Equation 1, as written here and by others (2, 3, 6), implies that poly-
nucleotide phosphorylase catalyzes the formation of polynucleotide chains
from mononucleotides alone.  Assuming this mechanism, it might be ex-
pected that in the reverse direction, namely phosphorolysis, cleavage would
result in the complete breakdown of a polynucleotide to mononucleotide
units.  The data presented above demonstrate that this is not the case.
The phosphorolysis of oligonucleotides containing 3 or 4 nucleoside residues
results in the accumulation of the compounds with 2 nucleoside units.  In
confirmation, Tables I and I1 show that pApA, ApA, and ApU are not phos-
phorolyeed at significant rates.

One possible explanation for these results may be that the enzyme
demonstrates an exacting specificity when the substxate presented to it has
only 2 nucleoside units.  The condensation of 2 ADP molecules according
to Equation 1 would be expected to form ppApA, a dinucleotide with a
pyrophosphate end group.  This compound has not yet been prepared, and
it might undergo phosphorolysis.

An alternative conclusion could be that polynucleotide phosphorylase,
like starch phosphorylase (26) , catalyzes only a limited phosphorolysis of
the chain, and the "limit oligon~cleotide'~ happens to be a dinucleotide.
Recent experiments (27,28) have suggested that purified preparations of A.
winehndii polynucleotide phosphorylase catalyze the condensation of 2
mononucleotide units to a dinucleotide very slowly, if at all.  Thus, a lag
in the polymerization reaction can be overcome by the addition of a pre-
formed polynucleotide chain to the reaction mixture.  In analogy with the
enzymic synthesis of polysaccharides, the new polymer has been shown to
be built onto this primer.  It is consistent with the experiments reported
here that the primer may be as small as a dinucleotide (28).

M. F. SINGER 227

The author wishes to thank Dr. Leon A. Heppel for suggesting this
problem and for many stimulating discussions during the course of the
work.

SUMMARY

The phosphorolysis of oligonucleotides by preparations of polynucleotide
phosphorylase from Azotobacter winehndii and Escherichia coli has been
studied.  Tri- and tetranucleotides with a phospho monoester group at the
terminal C-5' were readily phosphorolyzed; however, if the phospho
monoester group was at the terminal C-3', the compounds were resistant
to enzymic attack.  Trinucleoside diphosphates and tetranucleoside tri-
phosphates were also phosphorolyzed by these enzymes, indicating that the
C-5' phospho monoester moiety is not required in order that an oligonucleo-
tide be a substrate.  Dinucleotides and dinucleoside monophosphates were
not phosphorolyzed at significant rates.  The products of the phosphoro-
lysis of the compounds with 3 and 4 nucleoside residues were identified as
the nucleoside diphosphates (ADP or UDP) and the resistant dinucleotide
or dinucleoside monophosphate.

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