Tài liệu Báo cáo Y học: Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase ppt

Dinucleoside polyphosphates stimulate the primer independent
synthesis of poly(A) catalyzed by yeast poly(A) polymerase
Marı
´
aA.Gu¨ nther Sillero, Anabel de Diego, Hugo Osorio and Antonio Sillero
Departamento de Bioquı
´
mica, Instituto de Investigaciones Biome
´
dicas Alberto Sols UAM/CSIC, Facultad de Medicina,
Madrid, Spain
Novel properties of the primer independent synthesis of
poly(A), catalyzed by the yeast poly(A) polymerase are
presented. The commercial enzyme from yeast, in contrast to
theenzymefromEscherichia coli, is unable to adenylate the
3¢-OH end of nucleosides, nucleotides or dinucleoside poly-
phosphates (Np
n
N). In the presence of 0.05 m
M
ATP,
dinucleotides (at 0.01 m
M
) activated the enzyme velocity in
the following decreasing order: Gp
4
G, 100; Gp
3
G, 82; Ap
6
A,

61; Gp
2
G, 52; Ap
4
A, 51; Ap
2
A, 41; Gp
5
G, 36; Ap
5
A, 27;
Ap
3
A, 20, where 100 represents a 10-fold activation in
relation to a control without effector. The velocity of the
enzyme towards its substrate ATP displayed sigmoidal kin-
etics with a Hill coefficient (n
H
)of1.6andaK
m
(S
0.5
) value of
0.308 ± 0.120 m
M
. Dinucleoside polyphosphates did not
affect the maximum velocity (V
max
) of the reaction, but did
alter its n

H
and K
m
(S
0.5
) values. In the presence of 0.01 m
M
Gp
4
GorAp
4
Athen
H
and K
m
(S
0.5
) values were (1.0 and
0.063 ± 0.012 m
M
) and (0.8 and 0.170 ± 0.025 m
M
),
respectively. With these kinetic properties, a dinucleoside
polyphosphate concentration as low as 1 l
M
may have a
noticeable activating effect on the synthesis of poly(A) by the
enzyme. These findings together with previous publications
from this laboratory point to a potential relationship

between dinucleoside polyphosphates and enzymes catalyz-
ing the synthesis and/or modification of DNA or RNA.
Keywords:Ap
4
A; Gp
4
G; dinucleoside polyphosphates; yeast
poly(A) polymerase.
We have recently shown that Escherichia coli poly(A)
polymerase adenylates the 3¢-OH end of nucleosides,
nucleotides and dinucleotides of the type nucleoside (5¢)
oligophospho (5¢) nucleosides (Np
n
N¢) [1]. This novel
property of E.colipoly(A) polymerase moved us to analyze
whether these compounds were also substrates of eukaryotic
yeast poly(A) polymerase. The yeast enzyme is involved in
the processing of the 3¢-OH end of mRNA [2,3], forming a
complex with two cleaving factors and a polyadenylation
factor [4,5]. The core yeast poly(A) polymerase appears to
have a molecular mass of around 63 kDa [3]. Separated
form the complex, the core yeast enzyme catalyzes the
addition of poly(A) tails to a variety of RNAs or poly(A) of
different lengths [3]. The experiments described below were
carried out with a commercial preparation obtained from an
E.coli strain containing a clone of the yeast poly(A)
polymerase gene [6]. In principle, it can be assumed that this
preparation corresponds to pure poly(A) polymerase with
no contaminating cleaving factors.
While using this preparation, we observed that primer

independent poly(A) synthesis was activated by dinucleo-
side polyphosphates. The findings reported here could open
new views both on the catalytic properties of yeast poly(A)
polymerase and on the intracellular role of dinucleoside
polyphosphates, a family of compounds of increasing
metabolic and regulatory interest [7–11].
MATERIALS AND METHODS
Materials
Poly(A) polymerase from yeast was from Amersham
Pharmacia Biotech (Code 74225Z, lot numbers: 109217;
109899; 110278; 111182. One unit of enzyme is the
amount that incorporates 1 nmol of ATP (as AMP) into
an acid insoluble form in 1 min at 37 °C. These
preparations contained 761 UÆmL
)1
(1522 UÆmg
)1
pro-
tein). When required, the enzyme was diluted in 0.25%
bovine serum albumin (BSA). Shrimp alkaline phospha-
tase (EC 3.1.3.1) was from Roche Molecular Biochemicals
and phosphodiesterase (from Crotalus durissus,EC
3.1.4.1) was from Boehringer Mannheim. [a-
32
P]ATP
(3000 CiÆmmol
)1
) was from Dupont NEN. TLC silica-gel
fluorescent plates were from Merck. X-ray films were
from Konica Corporation. Radioactively labeled nucleo-

tides were quantified by the use of an InstantImager
(Packard Instrument Co.) HPLC was carried out in a
Hewlett Packard chromatograph (model 1090), with a
diode array detector, commanded by an HPLC Chem-
Station. The Hypersil ODS column (2.1 · 100 mm) was
from Hewlett Packard.
Correspondence to A. Sillero, Departamento de Bioquı
´
mica,
Facultad de Medicina UAM, C/Arzobispo Morcillo 4, 28029
Madrid, Spain. Fax: + 34 91 5854587, Tel.: + 34 91 3975413,
E-mail:
Abbreviations:Gp
n
G, guanosine(5¢)oligophospho(5¢)guanosine;
Np
n
N, nucleoside (5) oligophospho (5¢) nucleosides.
Enzymes: alkaline phosphatase (EC 3.1.3.1); phosphodiesterase from
Crotalus durissus (EC 3.1.4.1); poly(A) polymerase from Escherichia
coli and from yeast (EC 2.7.7.19).
(Received 9 July 2002, revised 9 September 2002,
accepted 11 September 2002)
Eur. J. Biochem. 269, 5323–5329 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03253.x
METHODS
Enzyme assays
Unless indicated otherwise the reaction mixtures con-
tained 20 m
M
Tris/HCl pH 7.0, 50 m

M
KCl, 0.7 m
M
MnCl
2
,0.2m
M
EDTA, 100 lgÆmL
)1
acetylated bovine
serum albumin (acetylated BSA), 10% (v/v) glycerol,
ATP, MgCl
2
, yeast poly(A) polymerase and, where
indicated, other nucleotides or dinucleotides. After incu-
bation at 30 °Cor37°C the reaction mixtures were
analyzed by TLC or HPLC. When indicated, the
reaction mixtures were treated with 20 UÆmL
)1
shrimp
alkaline phosphatase for 1 h at 37 °C, and after inacti-
vation of the phosphatase, by heating at 90 °Cfor
5 min, treated further with 20 lgÆmL
)1
phosphodiesterase
for 1 h at 37 °C.
TLC
The reaction mixtures (usually 0.01–0.02 mL) contained
(0.02 m
M

)[a-
32
P]ATP (20 lCiÆmL
)1
). Aliquots (0.0015 mL)
of the reaction were taken, spotted on silica gel plates, and
developed in dioxane/ammonium hydroxide/water 6 : 1 : 6
(v/v/v). Nucleotide spots were localized with a 253-nm
wavelength light and the radioactivity measured by auto-
radiography and/or with an InstantImager.
HPLC
Aliquots (0.01 mL) of the reaction mixtures (usually in a
volume of 0.035 mL) were transferred into 0.1 mL of water
andkeptat95°C for 1.5 min. After chilling, the mixtures
were filtered (using a Millipore HA, 0.45 lm nitrocellulose
membrane) and a 0.05-mL aliquot injected into a Hypersil
ODS column. Elution was performed at a flow rate of
0.5 mLÆmin
)1
with a 20-min linear gradient (5–30 m
M
)of
sodium phosphate (pH 7.5), in 20 m
M
tetrabutylam-
monium bromide/20%methanol (v/v) (buffer A) followed
by a 10-min linear gradient (30–100 m
M
) of sodium
phosphate (pH 7.5) in buffer A.

RESULTS
Comparison of poly(A) polymerase from
E. coli
and yeast
As stated in the Introduction, E.colipoly(A) polymerase,
in the presence of micromolar concentrations of ATP,
adenylates the 3¢-OH residues of most of the nucleosides,
nucleotides and dinucleotides tested and, under our
experimental conditions, is unable to catalyze the synthesis
of a poly(A) chain in the absence of a primer [1]. In order
to explore whether the yeast enzyme also exhibited the
same properties we assayed, in parallel, the activity of
both enzymes on guanosine, GDP and diguanosine
tetraphosphate (Gp
4
G), in the presence of 0.02 m
M
[a-
32
P]ATP. While confirming the adenylylation of these
compounds and the absence of synthesis of poly(A) by the
E.colipoly(A) polymerase, we did not observed adenyly-
lation of guanosine, GDP or Gp
4
G by the yeast enzyme.
In the absence or presence of these compounds, labeled
ATP was transformed mainly into a radioactive spot
retained at the origin of the TLC plate, a position that
could correspond to poly(A) chain(s). In addition, the
chromatographic pattern of the radioactive synthesized

products changed in the presence of Gp
4
G(seebelow).
From these results, it seemed of interest to explore the
effect of diguanosine polyphosphates (Gp
n
G) on yeast
poly(A) polymerase.
Effect of diguanosine polyphosphates on yeast poly(A)
polymerase
The enzyme was incubated with of 0.2 m
M
ATP and in the
absence or presence of Gp
2
G, Gp
3
G, Gp
4
Gandthe
reaction products analyzed by HPLC after 30, 60 and
120 min incubation. In the absence of dinucleotides, the
amount of ATP decreased slowly along the incubation time,
with no concomitant increase of any ATP derivative
(Fig. 1A) In the presence of diguanosine polyphosphates
(Gp
2
G, Gp
3
GorGp

4
G), ATP consumption was strongly
stimulated, but again, formation of potential products of the
reaction was not observed. The results obtained after
30 min incubation are represented in Fig. 1B. The apparent
loss of ATP was assumed to be due to the formation of a
product, probably poly(A), that could be retained by the
column.
To test this assumption, the enzyme was incubated with
0.2 m
M
ATP, under the same experimental conditions as
in Fig. 1, for 60 min at 37 °C. A control without enzyme
was also carried out. The complete reaction mixture was
then divided into equal parts and one of them treated
with phosphodiesterase. The three samples involved were
analyzed by HPLC. The amount of ATP in the control,
indicates the ATP present at the start of the reaction
(Fig. 2A); the ATP that was consumed after incubation
with the polymerase (Fig. 2B), was totally recovered as
AMP (Fig. 2C), when the reaction mixture was treated
with phosphodiesterase before analysis by HPLC. From
these results (Figs 1 and 2), it can be concluded that
poly(A) was synthesized from ATP, in the absence of
primer, and that Gp
2
G, Gp
3
G, and Gp
4

G stimulated that
synthesis.
Stimulation of poly(A) synthesis as a function of
diguanosine diphosphate (Gp
2
G) concentration
The concentration of dinucleoside polyphosphate needed
to stimulate the synthesis of poly(A) was analyzed using
Gp
2
G as effector. Yeast poly(A) polymerase was incu-
batedwith0.02m
M
[a-
32
P]ATP, in the absence and
presence of three different concentrations of Gp
2
G
(0.001, 0.010, or 0.050 m
M
). After 5, 10 and 20 min
incubation, aliquots of the reaction mixture were
analyzed by TLC. The results corresponding to the
5-min incubation are shown in Fig. 3. No appreciable
synthesis of poly(A) (spot at the origin) was observed in
the absence of Gp
2
G, whereas in its presence the ATP
spot decreased, increasing concomitantly the radioactivity

at the origin. In the presence of 0.01 or 0.050 m
M
Gp
2
G,
almost no ATP was left in the assay after 5 min
incubation. These results show that a concentration as
low as 0.001 m
M
Gp
2
G stimulates, under these condi-
tions, the synthesis of poly(A) catalyzed by yeast poly(A)
polymerase around sixfold.
5324 M. A. Gu
¨
nther Sillero et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Relative activity of Gp
n
Gs as effectors of the synthesis
of poly(A)
Based on the above results, the effect of several diguanosine
polyphosphates on the synthesis of poly(A) was comparat-
ively studied. The enzyme was incubated for 10 min with
0.05 m
M
[a-
32
P]ATP, and in the absence or presence of
Gp

2
G, Gp
3
G, Gp
4
G, Gp
5
G (0.01 m
M
each). Aliquots of
the reaction mixture were applied to a TLC plate. Under
these conditions, the maximum ATP consumed was less
than 50% (Fig. 4A). Formation of poly(A) (spots at the
origin) was clearly seen in the samples containing dinucleo-
tides, but scarcely visible in the control reaction (with
enzyme and without Gp
n
G) carried out in duplicate (lanes
C). The reaction mixtures were treated further with alkaline
phosphatase and (after inactivation of the phosphatase)
with phosphodiesterase and analyzed by TLC as above
(Fig. 4B). The results round off those presented in Figs 4A
i.e. AMP, representing the amount of ATP incorporated
into poly(A), appears preferentially in the reaction mixtures
containing the effectors (Fig. 4B). From the radioactivity
present in the AMP spot, the relative capacity of diguan-
osine polyphosphates to stimulate the synthesis of poly(A),
considering a media of four experiments, was: Gp
4
G, 100;

Gp
3
G, 82; Gp
2
G, 52; Gp
5
G, 36, where 100 represents a
10-fold activation in relation to a control without effector.
Effect of diadenosine polyphosphates on poly(A)
polymerase
Previous experiments had shown that diadenosine poly-
phosphates also stimulated the synthesis of poly(A)
catalyzed by yeast poly(A) polymerase. The relative
activity of diadenosine polyphosphates as effectors of
the poly(A) synthesis was assayed as in Fig. 4, using
0.05 m
M
[a-
32
P]ATP as substrate, in the absence or
presence of 0.01 m
M
Ap
n
As. The relative efficiency of
diadenosine polyphosphates to stimulate the synthesis of
poly(A), considering a media of four experiments, was:
Ap
6
A, 61; Ap

4
A, 51; Ap
2
A, 41; Ap
5
A, 27; Ap
3
A, 20
(results not shown). These values were calculated relative
to the maximal activation (100) considered for Gp
4
G(see
above).
Dinucleoside polyphosphates diminish the K
m
(S
0.5
) value
for ATP in the primer independent synthesis of poly(A)
In order to understand why dinucleoside polyphosphates
activated the primer independent synthesis of poly(A), the
effect of 0.01 m
M
Gp
4
GorAp
4
A on the synthesis of
poly(A) was analyzed at different ATP concentrations
(0, 0.025, 0.05, 0.1 and 0.2 m

M
). Samples were taken after
10 min incubation (a time at which the velocity of the
reactions were linear, as tested in previous assays) spotted
on TLC plates and the rate of synthesis of poly(A) as a
function of ATP concentration determined as in Fig. 4.
Moreover, in these conditions less than 30% of the ATP
was consumed in the case of the reaction mixtures
containing effectors and the lowest concentration of
substrate. The Michaelis-Menten (Fig. 5A), Lineweaver-
Burk (Fig. 5B) and Hill (Fig. 5C) plots of the results
showed that the enzyme presented a sigmoidal kinetics
that tended to hyperbolic in the presence of Gp
4
Gor
Ap
4
A. From these plots, maximum velocity (V
max
)and
K
m
(S
0.5
) values were determined. In the absence of
effector, the enzyme presented a Hill coefficient of around
1.6 that decreased to around 1.0 and 0.8 in the presence
of 0.01 m
M
Gp

4
GorAp
4
A, respectively. The K
m
(S
0.5
)
Fig. 1. Effect of diguanosine polyphosphates
(Gp
2
G, Gp
3
G, Gp
4
G) on the consumption of
ATP catalyzed by yeast poly(A) polymerase.
The reaction mixtures (0.035 mL) contained:
20 m
M
Tris/HCl, pH 7.0, 50 m
M
KCl, 0.7 m
M
MnCl
2
,0.2m
M
EDTA, 100 lgÆmL
)1

acetyl-
ated BSA, 10% glycerol, 0.5 m
M
MgCl
2
,
0.2 m
M
ATP and 0.44 units of the enzyme
(part A). Reaction mixtures supplemented
with 0.04 m
M
Gp
2
G, 0.1 m
M
Gp
3
GorGp
4
G
are shown in part (B) of the figure. After 30, 60
and 120 min incubation at 37 °C, aliquots
were taken and analyzed by HPLC as indica-
tedinMaterialsandmethods.
Ó FEBS 2002 Poly(A) polymerase activation by dinucleotides (Eur. J. Biochem. 269) 5325
values for ATP were 0.308 ± 0.120 m
M
(n ¼ 5),
0.063 ± 0.012 m

M
(n ¼ 3) and 0.170 ± 0.025 m
M
(n ¼ 3) in the absence or presence of Gp
4
GorAp
4
A,
respectively. The V
max
value determined for the primer
independent synthesis of poly(A) was about the same in
the absence or presence of dinucleotides, i.e. around
500 UÆmL
)1
[equivalent to a rate (k
cat
)ofAMPincor-
poration of 1 s
)1
] a value close to that determined in the
presence of poly(A) as a primer, as stated by the
manufacturer.
DISCUSSION
Some experimental aspects can be considered firstly, in
relation to the methods currently used by others to assay
poly(A) polymerases. As noted previously [1], the labeled
RNA-(A)
n
products synthesized by polymerases are usually

determined by acid precipitation or phenol extraction and
ethanol precipitation. The amount of radioactivity deter-
mined in those precipitates is the parameter used to
determine the poly(A) polymerase activity [12–18]. Potential
reaction products that do not precipitate with these
procedures may pass unnoticed.
Adenylation of nucleosides, nucleotides and dinucleotides
by E.colipoly(A) polymerase [1] was detected using TLC
and HPLC methods, the same two methods used in this
work to study the yeast enzyme. The TLC procedure
involves spotting aliquots of the complete reaction mixture
onto a plate and analysis of all the potential reaction
products synthesized during incubation. In the HPLC
procedure, the reaction mixture is heated and filtered (see
Materials and methods). All the poly(A) products synthes-
ized from ATP passed through this filter, but were retained
by the precolumn or column. The enzyme activity could be
followed either, by measuring the decrease of the ATP
content in the reaction mixture or by treating the reaction
mixture first with alkaline phosphatase (to hydrolyze
residual adenosine 5¢-phosphates to adenosine) and then
with phosphodiesterase to hydrolyze the synthesized
poly(A) to AMP. According to our results, the amount of
Fig. 2. ATP consumption catalyzed by yeast poly(A) polymerase. The
reaction mixtures (0.035 mL) contained: 20 m
M
Tris/HCl, pH 7.0,
50 m
M
KCl, 0.7 m

M
MnCl
2
,0.2m
M
EDTA, 100 lg/mL acetylated
BSA, 10% glycerol, 0.5 m
M
MgCl
2
,0.2m
M
ATP and in the absence
(A) or presence of 0.76 units of the enzyme (B and C). After 60
incubation at 37 °C (B) an aliquot of the reaction mixture was then
treated further with phosphodiesterase (C). The analysis was per-
formed by HPLC as indicated in Materials and methods. The areas of
the peaks corresponding to ATP (A and B) and AMP (C) were, in
arbitrary units, 1243, 175 and 1265, respectively.
Fig. 3. Effect of different concentrations of Gp
2
G on the synthesis of
poly(A) catalyzed by yeast poly(A) polymerase. The reaction mixture
(0.01 mL) contained: 0.02 m
M
ATP, 0.2 lCi [a-
32
P]ATP, Gp
2
G(as

indicated), 0.38 units of the enzyme and other conditions as described
in Materials and methods. After 5 min incubation at 37 °C, aliquots
were taken and analyzed by TLC. Lane (– E): control without enzyme.
5326 M. A. Gu
¨
nther Sillero et al. (Eur. J. Biochem. 269) Ó FEBS 2002
AMP so obtained was equimolar to the ATP consumed
during the enzyme reaction.
ThedifferencebetweentheenzymefromE.coliand yeast
concerning their substrate specificity towards nucleosides,
nucleotides and dinucleotides is also worth noting. The
yeast poly(A) polymerase, contrary to the E.colienzyme, is
apparently unable to adenylate the 3¢-OH end of those
compounds. However, the primer independent activity of
the yeast enzyme is strongly activated by dinucleoside
polyphosphates. Commercial yeast poly(A) polymerase
presented, in the absence of primer, a sigmoidal kinetics
towards its substrate ATP, with a Hill coefficient (n
H
)of
around 1.6. The presence of Gp
4
GorAp
4
Achangedthe
kinetic from sigmoidal to hyperbolic, decreasing the K
m
(S
0.5
) value from 0.308 ± 0.120 m

M
to 0.063 ± 0.012 m
M
and 0.170 ± 0.025 m
M
in the presence of Gp
4
GorAp
4
A,
Fig. 4. Effect of diguanosine polyphosphates (Gp
2
G, Gp
3
G, Gp
4
G,
Gp
5
G) on the synthesis of poly(A) catalyzed by yeast poly(A) poly-
merase. The reaction mixture (0.02 mL) contained: 0.05 m
M
ATP,
0.4 lCi [a-
32
P]ATP, 0.01 m
M
Gp
n
G, 0.19 units of the enzyme and

other conditions as described in Materials and methods. After 10 min
incubation at 30 °C, aliquots were taken, and spotted on a TLC plate
(Part A). The rest of the reaction mixture was treated with shrimp
alkaline phosphatase and phosphodiesterase as described in Materials
and Methods and analyzed as above (Part B). Lane (– E): control
without enzyme; lanes (C): complete reaction with no added dinu-
cleotide; other lanes (1–4) with added Gp
n
G(0.01m
M
)asindicated.
Fig. 5. Influence of ATP concentration on the primer independent syn-
thesis of poly(A) catalyzed by yeast poly(A) polymerase. EffectofAp
4
A
or Gp
4
G. The reaction mixture (0.02 mL) contained: variable
concentrations of [a-
32
P]ATP (0.025–0.2 m
M
) specific activity:
320 lCiÆlmol
)1
,0.2m
M
MgCl
2
, 0.1 units of the enzyme, 0.01 m

M
Ap
4
AorGp
4
G where indicated and other conditions as described in
Materials and methods. After 10 min incubation at 30 °C, the reaction
was stopped by heating 2 min at 90 °C, treated with alkaline phos-
phatase and phosphodiesterase and analyzed by TLC. (v,isexpressed
as lmol of AMP incorporated min
)1
ÆmL
)1
).
Ó FEBS 2002 Poly(A) polymerase activation by dinucleotides (Eur. J. Biochem. 269) 5327
respectively. This would result in an activation of the
polymerase at low ATP concentrations.
We have as yet no clue on either the mechanism of action
or the physiological significance of these findings. Never-
theless, the following points could be raised.
TheoccurrenceofAp
4
A, at (sub)micromolar concentra-
tions has been described in all prokaryotic and eukaryotic
systems examined [19] and the presence of millimolar
concentrations of Gp
3
GandGp
4
GinArtemia and other

crustacea is well documented [20,21]; in addition, many
members of the Gp
n
G, Ap
n
AandAp
n
G families have been
found in human blood platelets [22,23].
About the presence of these compounds in the nucleus,
Ap
4
A is a dinucleotide specifically described to be present
in that organelle [24,25], but due to the pore size of the
nuclear envelope it can be considered that the (di)nucleo-
tide content in the nucleus may be similar to that in whole
cells [26]. An additional, and still unsolved problem, is the
question of how much of the (di)nucleotide content in
nuclei is free or ligated to nuclear structures [27] or
present in the environment in which the poly(A) poly-
merase is located. This may have an influence on the
enzyme as it seems to be a relationship between the
enzyme activity and the concentration of both ATP and
dinucleotides: poly(A) polymerase displays a sigmoidal
kinetics that becomes hyperbolic in the presence of
dinucleotides, a behavior that greatly enhances the enzyme
activity particularly at low ATP concentrations; for
instance at 0.02 m
M
ATP, concentrations as low as

1 l
M
of some dinucleotides may increase poly(A) synthe-
sis more than sixfold.; the influence that this activation
could have on the processing of the 3¢-OH end of mRNA
could also be considered.
The sigmoidal kinetics displayed by the enzyme favors the
view that poly(A) polymerase may contain an allosteric area
for a dinucleotide or (a dinucleotide-like structure) with the
following apparent preferences: comparing dinucleotides
with the same number of inner phosphates, guanine
dinucleotides are more active than adenine dinucleotides
and, adenine dinucleotides with even number of inner
phosphates tend to be more efficient than those with odd
number of phosphates.
We are aware that poly(A) polymerase has been
described as a multienzyme complex that may have in vivo
different, or additional, properties to those reported here. In
any event, yeast poly(A) polymerase, as supplied by the
manufacturer, is strongly activated by lmolar concentra-
tions of dinucleotides, preferentially at low ATP concen-
trations. The physiological significance of these findings
deserves further exploration.
ACKNOWLEDGEMENTS
This investigation was supported by grants from Direccio
´
nGeneralde
Investigacio
´
nCientı

´
ficayTe
´
cnica (PM98/0129; BMC2002-00866) and
Comunidad de Madrid (08/0021.1/2001). H.O was supported by a
Fellowship from Fundac¸ a
˜
oparaaCieˆ ncia e a Tecnologia (SFRH/BD/
1477/2000).
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nucleoside 5¢-phosphates and nucleoside (5¢) oligophospho (5¢)
nucleosides (Np
n
N). Eur. J. Biochem. 268, 1–8.
2. Haff, L.A. & Keller, E.B. (1975) The polyadenylate polymerases
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