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T7 DNA polymerase is not a zinc-metalloenzyme and the polymerase and exonuclease activities are inhibited by zinc ions
Vol. 122, No. 3, 1984
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 16, 1984
Pages 1410-1417
T7 DNA POLYMERASE IS NOT A ZINC-METALLOENZYME AND THE POLYMERASE AND EXONUCLEASE ACTIVITIES ARE INHIBITED BY ZINC IONS Ivan
Slaby**,
Birger
Department of Chemistry Karolinska Institutet, Received
July
I,
Lind"
and Arne
Holmgren
and *Department of Environmental Box 60400, S-104 01 Stockholm,
Hygiene, Sweden
11, 1984
Phage T7 DNA polymerase purified to homogeneity by an antithioredoxin immunoadsorbent technique was resolved into its active subunits the gene 5 protein and Escherichia coli thioredoxin by a novel technique involving chromatography on SephadexG-50 at pH 11.5. Analysis of the metal content of the holoenzyme by atomic absorption spectroscopy showed that it did not contain stoichiometric amounts of zinc. Determination of polymerase and exonuclease activities of the holoenzyme and the gene 5 protein in assay mixtures containing enzyme concentrations in excess of the Zn*+ concentrain no stimulation and tion showed full activity. Addition of Zn2+ resulted the activities were completely inhibited by 0.1 mM Zn*+. These results demonstrate that the essential T7 DNA polymerase is not a zinc-metalloenzyme and suggest that DNA polymerases show no functional requirement for Zn*+
DNA polymerases In addition, activity
they
require
--in vitro.
polymerase fully
DNA polymerase
of two subunits
thioredoxin
(Mr 12.000)
has no DNA polymerase (9-11). the ties
Addition
(7,
is 8),the
in
I:1
activity,
5' to 3' DNA polymerase (9-11).
a role
amounts
a unique
virus
but
reports induced
and double-stranded A method
splitting
T7 DNA polymerase
in subunits
**Present Karlovarska
address: Department of Medical 48, Plzen, Czechoslovakia.
found
essential
coded
enzyme comand -__ E. coli gene 5 protein
3' to 5' exonuclease
gene 5 protein
will
3' to 5' exonuclease active
by gelchromatography
Chemistry,
in a
6) or in wild
(Mr 80.000)
to prepare
DNA
(l-3).
The phage
to the
were
(5,
is a single-stranded
--in vitro
Zn *+ in the
of zinc
T7 gene 5 protein stoichiometry.
for
coli
to previous
(l-4).
Mg*+ or Mn2+ , for
either
I of Escherichia
in contrast
of thioredoxin
of the holoenzyme
have questioned
DNA polymerase I (6)
as Zn-metalloenzymes
cation,
no stoichiometric
Phage T7,DNA polymerase posed
regarded
divalent
results
since
cloned
been
an added
Recent
reaction
active
type
have generally
Charles
induce activi-
gene 5 protein
by
in 6 M guanidine-
University,
Vol. 122, No. 3, 1984
BIOCHEMICAL
HCl has been described a series
of dialysis
has generally (11,
steps
been assumed
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Renaturation of the gene 5 protein 2+ containing buffer (ll), against Zn that
T7 DNA polymerase
is made by since
it
is a Zn- metalloenzyme
12). We have
previously
merase
to homogeneity
graphy
(13,
merase
for
resolve
(11).
AND
14). its
developed based
the enzyme
into
investigation
and also its
method
on antithioredoxin
In the present zinc-content
a simple
developed
to purify
T7 DNA poly-
immunoadsorbent we have examined a novel
chromatoT7 DNA poly-
and simple
method
to
subunits.
MATERIAL AND METHODS E. co11 B/l was obtained from Dr. C.C. Richardson and bacteriophage T73 6 mutant from Dr. F. W. Studier. dATP, dCTP, dGTP and salmon sperm DNA we& purchased from Sigma. dTTP was from P-L Biochemicals, and(3H) dTTP and (methyl-3H )thymidine were from Radiochemical Centre, Amersham. Ultrapure MgC12, ZnC12, EDTA (Titriplex III) and Tris were from Merck, dithiothreitol (DTT) from Calbiochem, Sephadex G-25, G-50 and Sepharose 4B from Pharmacia Fine Chemicals and Chelex-100 (200-400 mesh, Na-form) from Bio-Rad laboratories. Horse liver alcohol dehydrogenase was a gift from Dr. C.-I. Branden, Uppsala, Sweden. Human carbonic anhydrase was a gift from Dr. S. Lindskog, Umeb, Sweden. T7 DNA polymerase was purified to homogeneity from -7 E. coli B/l cells infected with bacteriophage T73 6 with a technique based on immunoadsorbent affinity chromatography (13, 141. The enzyme binds to a column of anti-thioredoxin Sepharose 4B and is eluted in fully active form by a pulse of buffer at pH 11.5. A final phosphocellulose chromatography step yields T7 DNA polymerase of more than 99% purity (13, 14). Gene 5 protein was prepared from purified T7 DNA polymerase by separation from thioredoxin as follows. 2.5 nmole of T7 DNA polymerase was incubated for 10 min at 24OC in 500 pl of buffer at pH 11.5 (0.1 M glycin-NaOH, 0.5 M NaCl, 0.1 mM EDTA, 1 mM DTT). The mixture was then applied to a column of Sephadex G-50 (0.7 x 46 cm) equilibrated with the same buffer at 4oC. Fractions of about 0.6 ml were collected and neutralized with 1.0 M Tris-Cl buffer, pH 7.0. T7-(3H)-DNA was prepared by infection of E. coli B/l with bacteriophage T7 wild type in the presence of (methyl-3H )th@iid?iie (7). After the extraction with pher,ol , T7-(3H)-DNA was further purified on a column of Sepharose 4B (0.7 x 46 cm) in 10 mM Tris-Cl, pH 7.5, 0.2 M NaCl, 2 mM EDTA. Fractions from the chromatogram corresponding to high molecular weight DNA were pooled and used in exonuclease activity determinations. The specific activity of the T7-(3H)-DNA was 6 cpm/pmol of phosphorus equivalents. DNA polymerase activity was determined by a modification of the procedure described by Modrich and Richardson (7). The assay mixture (0.15 ml) contained 93 mM Tris-Cl H 7.6, 2.5 mM DTT, 10 mM MgC12, 53 mM NaCl, 0.15 mM dATP, dGTP, dCTP, and(St H)dTTP (2-5 cpm/pmol), 0.3 mg/ml of bovine serum albumin and 0.5 mM heat-denaturated salmon sperm DNA. The incubations were carried out for 30 min at 370C or 2 min at 17oC. One unit of DNA polymerase catalyzes the incorporation of 10 nmol of total nucleotide into an acidinsoluble product in 30 min at 370C (7). Exonuclease activities were assayed in the mixture of 100 mM Tris, pH 7.5, 10 mM MgC12, 2.5 mM DTT and 2.2 nmol of either native or heat-denaturated T7-(3H)-DNA in a final volume of 0.1 ml as described previously (13). The incubation time was 30 min at 37OC. One unit of exonuclease activity 1411
Vol. 122, No. 3, 1984
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE THE
ELECTROTHERMAL
I
ATOMIZATION
Temperature
PROGRAMME
Ramp
OC
time set
Hold
time set
Drying
100
5
20
Drying
200
5
10
Charring
400
6
24
Atomization
2 300
6
5
Cleaning
2 700
1
2
Cooling
20
15
5
Cooling
20
1
0
catalyzes the formation of 10 nmol of total acid-soluble nucleotide in 30 min at 37OC. Thioredoxin was assayed with thioredoxin reductase and NADPH using DTNB as the electron acceptor as described elsewhere (15). The reduction of DTNB was followed at 412 nm. Protein concentration was determined either by reading the absorbance at 280 nm (13) or by amino acid analysis. T7 DNA polymerase or the gene 5 protein samples were lyophilized and hydrolyzed with 6 M HCl - 0.5% phenol for 24 hours at IlOoC in vacua. Amino acid analysis was performed with a Beckman 121 M amino acxanalyzer. For the calculations,the total compositions of the gene 5 protein (16) and thioredoxin (13) were used. To remove loosely bound or contaminating zinc ions, T7 DNA polymerase and the standard metalloenzymes were applied to a Sephadex G-25 column before the zinc analysis. The column was prewashed with 5 mM EDTA (for 24 h) and equilibrated with 40 mM Tris-Cl buffer, pH 7.5. The equilibration took several days before a constant zinc background (5 rig/ml) was reached. In some experiments all the buffers and enzyme assay mixtures were passed through a column of Chelex-100 which had been converted to the magnesium form by washing with 1 M MgC12 (two bed volumes). Zinc was determined by atomic absorption spectrophotometry (AAS) at 213.8 nm (band-pass 0.7 nm) using an instrument (Perkin Elmer 373) equipped with an electrothermal atomization (ETA) unit (HGA-500), an automatic sample injector (AS-40), a printer (PRS-10)anda two-pen recorder (Model 56). A Zn hollow cathode lamp was used together with the deuterium background correction system. The ETA unit was programmed as shown in Table 1. The purging gas was 99.99% pure argon at an internal flow rate of 300 ml/ min (atomization step). This high gasflow combined with a long ramp time atomization was used to reduce the non-specific background signal to an acceptable level. Injections (20 ~1) of zinc standards or samples were made into standard graphite tubes. The areas under the atomization-signal peaks were integrated (11 s) by the instrument and used for evaluating the results after received on the printer. The recorder was used as a processcontroller with one pen recording the deuterium-compensated Zn signal and the other the non-specific background signal. All determinations were made in duplicate with at least two injections from each cup in AS-40. Zinc standards were prepared for each analysis by dilutions of a commercial standard solution with the buffer used in each experiment. All tips and vessels were carefully acid washed. Method of standard additions was used whenever possible. 1412
Vol.
122,
No.
BIOCHEMICAL
3, 1984
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Preparation 5 protein
and
graphy the
at
pH
of
mM EDTA, The
polymerase
or
exonuclease
(fig.
to
the
gene
5 protein
of of
2A).
G-25
of
zinc
the
bound
A peak
DNA polymerase ty
20,000
of of
zinc
was
peak
DNA polymerase
chromatography,
or was
while
the
separated
by
Sephadex
and
fractions 0.2
M NaCl,
the
the
of
less
to
of
peaks,
thioredoxin.
the
than had
but
zinc
DNA
content
equilibrated
pH protein
2::
was
residual
almost
T7
no
zinc.
DNA
In
zinc
changed
was buffer
followed
the
specific
after
decreased
the
Sephadex
substantial-
10
Effluent
Figure
1.
- Separation column to gene Gene 5 tivity tivity
(ml)
of the subunits of phage T7 DNA polymerase on a Sephadex G-50 at pH 11.5. The first peak corresponds 5 protein (e+) the second to thioredoxin (o------o). protein activity was measured as T7 3NA polymerase acafter complementation with thioredoxin; thioredoxin acwas followed as a reduction of DTNB (see Methods). of
1413
T7
activi-
1
5
two
order
polymerase
The
had
and
metal-free
activity.
content
polymerase
ly.
0
7.5,
a single-stranded
of
with
significantly
total
50X
purified
polymerase
not
protein
50 mM Tris-Cl,
- T7
for
observed,
two
chromato-
mg protein.
aliquot G-25
gene
G-50
second
and
polymerase
analyzed
the
about
activity
DNA
in
against
exonuclease per
DNA polymerase,
resulted
contained
an
Sephadex
T7
preparation
T7
zinc,
protein T7
in
of
5 protein
units
were
a column
gene
5 protein
double-stranded
loosely to
the
subunits
chromatography
1 mM DTT,
zinc-metallonezymes
applied
were The
gene
activity
remove
(fig.
I). to
Determination
- The
thioredoxin,
5'r0 glycerol,
recovered.
known
5 protein
corresponding dialysis
0.1
gene
E. coli -___
high
first
After
of
Vol. 122, No. 3, 1984
BIOCHEMICAL AND EGOPHYSICAL RESEARCH COMMUNICATIONS
r
L G
L
L)
n
0
”
L
Effluent
Figure
2.
of
symmetrical
genase
was
of
protein
to
have
the
of
standard in
Effect double-stranded creasing
T7
DNA of
carbonic
peak
ratio
the
found
human
found
zinc-protein and
*
0
ImU
- Chromatography of T7 DNA polymerase, preparation III (A) and human carbonic anhydrase (B) on a column of Sephadex G-25. The enzymes were applied to the column (0.8 x 20 cm) in 40 mM TrisCl buffer, pH 7.5. Fractions of 400 ~1 were collected and analyzed for the protein concentration ( M), the content of zinc (A---A) and DNA polymerase activity (M).
A sample one
b
.
the
anhydrase and
zinc
expected
three
applied (fig. zinc
2B).
content.
different
the
same
column
showed
Also
alcohol
Table
II
summarizes
T7
DNA
preparations No
zinc-metalloenzymes.
to
of
stoichiometric
dehydro-
amounts
the
polymerase of
Zn were
polymerase. zinc
and
EDTA
exonuclease concentrations
on
enzyme
activities of
zinc
activities were
to
the
assay
TABLE Zn ANALYSIS
OF 77 DNA POLYMERASE T7 DNA polymerase
Enzyme
inhibited mixtures
T7
DNA by (fig.
polymerase
addition 3A
AND THE STANDARD activity
B).
Zn content
dehydrogenase
3.9
Carbonic
anhydrase
1.1 prep.
I
10,000
0.27
prep.
II
13,000
0.17
prep.
III
10,600
0.35
1414
and
in-
ENZYMES
Alcohol
T7 DNA polymerase
of
II
g atoms/mole
units/mg
and
enzyme
The
Vol.
122,
No.
BIOCHEMICAL
3, 1984
AND
0 ZnCI,
Figure
same
3.
was gene
further
addition
hibition
was
veal of
any up
to Since
low
zinc
zinc-free mined
obtained
for
5 protein of reversed
effect
on
L
I
1
2
orEOTA
COMMUNICATIONS
Imtl,
the
single-stranded
(fig.
3'2).
The
magnesium
ions
to
by
addition
polymerase
of or
exonuclease
activities
were
the
assay
mixtures.
EDTA
(fig.
3).
exonuclease
activity
not
recovered
EDTA
the
itself in
the
by
However,
activities
of
did
in-
not
re-
concentration
2 mM. T7
DNA
polymerase
contamination enzyme.
with
RESEARCH
- The effect of ZnCl? and EDTA on DNA polymerase (A) and doublestranded DNA exonuclease (B) activities of T7 DNA polymerase and single-stranded DNA exonuclease activity of gene 5 protein (C). All three activities were measured in the presence of indicated amounts of EDTA (N) or ZnCl 2d (A+). The reactions of Zninhibited enzyme activities by a ditlon of EDTA are also shown (o----o): The enzyme was then preincubated for 10 min at 4'C in the assay mixtures containing 0.2 mM ZnC12 prior to addition of EDTA followed by the incubation for 30 min at 37OC. T7 DNA polymerase concentrations were 0.85 nM (A) and 1.28 nM (B), respectively. The concentration of gene 5 protein was 1.25 nM (C).
result
isolated
BIOPHYSICAL
higher
normally
present To
exclude
concentration
in this
is the
assay
possibility, of
T7
DNA
1415
assayed
at
mixture enzyme polymerase.
nM
concentrations,
could
a
reactivate
a
activity
was
By
a short
using
deterin-
Vol.
122,
No.
3, 1984
BIOCHEMICAL
AND
ZnCL,or
Figure
4.
cubation was
and
possible
100 fig.
to
without in
the
4,
decreasing
enzyme
EOTA lmMi
hibition
obtained amount
uM of
EDTA,
was
pretreated
form
and was
by
T7
DNA by
adding
incubation The
passage less by
of
the
polymerase.
unaffected
first
of
contained
addition
by
of
temperature
0.2
activity
inhibited
the
assay
magnesium
strongly
molar
COMMUNICATIONS
- The effect of ZnCl and EDTA on DNA polymerase activity at enzyme concentrate activity was measured .5 n. The polymerase the presence of EDTA (e--r ), ZnCl (LA) or EDTA and in equimolar amounts (o----o ). The 2 oncentration of T7 DNA meraie in the assay mixture was 0.2 PM and the incubation carried out for 2 min at 170C. The enzyme was first chromatographed through the column of zinc-free Sephadex G-25 and incubation mixture without DTA was treated by Chelex-100 magnesium form to remove Zn St ions.
time
mixture
BIOPHYSICALRESEARCH
additions
zinc.
As
zinc
was
in
uM
of
of
zinc.
Chelex-
As
excess
previous
shown
EDTA,
reversed
in
but
experiments,
partially
it
assay
a column
0.15
the in
17'C
polymerase
through than
to
high in ZnC12 polvwas -
the by
an
in-
equi-
EDTA.
DISCUSSION The
results
Sephadex
G-25
all
addition
of
functional
role
questioned
by
results
for
for Zn
2+
study
show
excess for the
in
results that
replication extending
activities Zn'+,
Zn
phage no
of
the
T7
DNA
enzyme
were
of
EDTA
addition
the
DNA
polymerase
reaction
for
DNA polymerase
I from
T7
essential DNA
generalization
(7,
virus-induced 8), that
metalloenz.ymes. 1416
polymerase
stoichiometric
whereas
the of
the
that
contained
enzyme
demonstrate
quired
this
chromatography,
Furthermore, by
of
shows DNA
amounts
of
strongly showed (1, E. -__
no
effect.
was (5,
A
recently 6).
Our
DNA polymerase
functional
polymerases
Zn.
inhibited
2)
coli
T7 no
after
re-
requirement are
not
Zn-
Vol.
122,
No.
We have 5 protein which
developed
based is
BIOCHEMICAL
3, 1984
used
on in
the
polymerase
enzyme,
the
gene
hibited
by
Zn2+
binding
of
Zn and
the
the
isolate
5 protein
a novel
thioredoxin
simple
stability
(13,
In to
14).
sulfhydryl contain
cases
T7
Similar
RESEARCH
to
DNA
affinity
single-stranded both
BIOPHYSICAL
technique of
immunoadsorbent
5 protein
ions
and
known
ions.
2+
AND
groups essential
isolate
polymerase
the at
chromatography to
the
T7
inhibition on
the sulfhydryl
protein.
pH,
method
be
was caused
Both groups
to holo-
activity may
gene
high
DNA polymerase
exonuclease this
COMMUNICATIONS
inby
the (12,
gene 17).
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. Barbro Sbderman and Mrs. Agneta Slaby is gratefully acknowledged. Ivan Slaby was supported by a long-term EM90 fellowship. This investigation was supported by grants from the Swedish Cancer Society, project 961, the Swedish Medical Research Council, 13X-3529 and the Knut and Alice Wallenberg Foundation. REFERENCES iI . Sorinqate, C.F., Mildvan, A.S., Abramson, R., Engle, J.L. and Loeb, L:A. (1973) J. Biol. Chem. 248, 5987-5993. Mildvan, A.S. and Loeb, L.A. (1979) CRC Critical Rev. Biochem. 6, 2. 219-244. Coleman, J.E. (1983) In: Zinc Enzymes, Ed. T.T. Spiro, John Wiley & 3. Sons, New York, pp. 221-252. Kornberg, A. (1980) DNA replication. W.H. Freeman & Co., San Francisco, 4. pp. l-724. Herrman, M.S. and Behnke, W.D. (1982) 5. Walton,K.E.,Fitzgerald, P.C., Biochem. Biophys. Res. Commun. 108, 1353-1361. 6. Ferrin, L.J., Mildvan, A.S. and Loeb, L.A. (1983) Biochem. Biophys. Res. Commun. 112, 723-728. 7. Modrich, P. and Richardson, C.C. (1975) J. Biol. Chem. 250, 5508-5522. 8. Mark, D.F. and Richardson, C.C. (1976) Proc. Natl. Acad. Sci. USA 73, 780-784. 9. Hori, K., Mark, D.F. and Richardson, C.C. (1979) J. Biol. Chem. 254, 11591-11597. IO. Hori, K., Mark, D.F. and Richardson, C.C. (1979) J. Biol. Chem. 254, 11598-11604. 11. Adler, S. and Modrich, P. (1979) J. Biol. Chem. 254, 11605-11614. 12. Adler, S. and Modrich, P. (1983) J. Biol. Chem. 258, 6956-6962. 13. Nordstrom, B., Randahl, H., Slaby, I. and Holmgren. A. (1981) 3. Biol. Chem. 256, 3113-3117. 14. Randahl, H., Slaby, I. and Holmgren, A. (1982) Eur. J. Biochem. 128, 445-449. 15. Slaby, I. and Holmgren, A. (1975) J. Biol. Chem. 250, 1340-1347. Dunn, J.J. and Studier, F.W. (1983) J. Mol. Biol. 166, 477-535. 167: Randahl, H. and Holmgren, A. (1984) J. Biol. Chem. submitted for publication.
1417
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 16, 1984
Pages 1410-1417
T7 DNA POLYMERASE IS NOT A ZINC-METALLOENZYME AND THE POLYMERASE AND EXONUCLEASE ACTIVITIES ARE INHIBITED BY ZINC IONS Ivan
Slaby**,
Birger
Department of Chemistry Karolinska Institutet, Received
July
I,
Lind"
and Arne
Holmgren
and *Department of Environmental Box 60400, S-104 01 Stockholm,
Hygiene, Sweden
11, 1984
Phage T7 DNA polymerase purified to homogeneity by an antithioredoxin immunoadsorbent technique was resolved into its active subunits the gene 5 protein and Escherichia coli thioredoxin by a novel technique involving chromatography on SephadexG-50 at pH 11.5. Analysis of the metal content of the holoenzyme by atomic absorption spectroscopy showed that it did not contain stoichiometric amounts of zinc. Determination of polymerase and exonuclease activities of the holoenzyme and the gene 5 protein in assay mixtures containing enzyme concentrations in excess of the Zn*+ concentrain no stimulation and tion showed full activity. Addition of Zn2+ resulted the activities were completely inhibited by 0.1 mM Zn*+. These results demonstrate that the essential T7 DNA polymerase is not a zinc-metalloenzyme and suggest that DNA polymerases show no functional requirement for Zn*+
DNA polymerases In addition, activity
they
require
--in vitro.
polymerase fully
DNA polymerase
of two subunits
thioredoxin
(Mr 12.000)
has no DNA polymerase (9-11). the ties
Addition
(7,
is 8),the
in
I:1
activity,
5' to 3' DNA polymerase (9-11).
a role
amounts
a unique
virus
but
reports induced
and double-stranded A method
splitting
T7 DNA polymerase
in subunits
**Present Karlovarska
address: Department of Medical 48, Plzen, Czechoslovakia.
found
essential
coded
enzyme comand -__ E. coli gene 5 protein
3' to 5' exonuclease
gene 5 protein
will
3' to 5' exonuclease active
by gelchromatography
Chemistry,
in a
6) or in wild
(Mr 80.000)
to prepare
DNA
(l-3).
The phage
to the
were
(5,
is a single-stranded
--in vitro
Zn *+ in the
of zinc
T7 gene 5 protein stoichiometry.
for
coli
to previous
(l-4).
Mg*+ or Mn2+ , for
either
I of Escherichia
in contrast
of thioredoxin
of the holoenzyme
have questioned
DNA polymerase I (6)
as Zn-metalloenzymes
cation,
no stoichiometric
Phage T7,DNA polymerase posed
regarded
divalent
results
since
cloned
been
an added
Recent
reaction
active
type
have generally
Charles
induce activi-
gene 5 protein
by
in 6 M guanidine-
University,
Vol. 122, No. 3, 1984
BIOCHEMICAL
HCl has been described a series
of dialysis
has generally (11,
steps
been assumed
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Renaturation of the gene 5 protein 2+ containing buffer (ll), against Zn that
T7 DNA polymerase
is made by since
it
is a Zn- metalloenzyme
12). We have
previously
merase
to homogeneity
graphy
(13,
merase
for
resolve
(11).
AND
14). its
developed based
the enzyme
into
investigation
and also its
method
on antithioredoxin
In the present zinc-content
a simple
developed
to purify
T7 DNA poly-
immunoadsorbent we have examined a novel
chromatoT7 DNA poly-
and simple
method
to
subunits.
MATERIAL AND METHODS E. co11 B/l was obtained from Dr. C.C. Richardson and bacteriophage T73 6 mutant from Dr. F. W. Studier. dATP, dCTP, dGTP and salmon sperm DNA we& purchased from Sigma. dTTP was from P-L Biochemicals, and(3H) dTTP and (methyl-3H )thymidine were from Radiochemical Centre, Amersham. Ultrapure MgC12, ZnC12, EDTA (Titriplex III) and Tris were from Merck, dithiothreitol (DTT) from Calbiochem, Sephadex G-25, G-50 and Sepharose 4B from Pharmacia Fine Chemicals and Chelex-100 (200-400 mesh, Na-form) from Bio-Rad laboratories. Horse liver alcohol dehydrogenase was a gift from Dr. C.-I. Branden, Uppsala, Sweden. Human carbonic anhydrase was a gift from Dr. S. Lindskog, Umeb, Sweden. T7 DNA polymerase was purified to homogeneity from -7 E. coli B/l cells infected with bacteriophage T73 6 with a technique based on immunoadsorbent affinity chromatography (13, 141. The enzyme binds to a column of anti-thioredoxin Sepharose 4B and is eluted in fully active form by a pulse of buffer at pH 11.5. A final phosphocellulose chromatography step yields T7 DNA polymerase of more than 99% purity (13, 14). Gene 5 protein was prepared from purified T7 DNA polymerase by separation from thioredoxin as follows. 2.5 nmole of T7 DNA polymerase was incubated for 10 min at 24OC in 500 pl of buffer at pH 11.5 (0.1 M glycin-NaOH, 0.5 M NaCl, 0.1 mM EDTA, 1 mM DTT). The mixture was then applied to a column of Sephadex G-50 (0.7 x 46 cm) equilibrated with the same buffer at 4oC. Fractions of about 0.6 ml were collected and neutralized with 1.0 M Tris-Cl buffer, pH 7.0. T7-(3H)-DNA was prepared by infection of E. coli B/l with bacteriophage T7 wild type in the presence of (methyl-3H )th@iid?iie (7). After the extraction with pher,ol , T7-(3H)-DNA was further purified on a column of Sepharose 4B (0.7 x 46 cm) in 10 mM Tris-Cl, pH 7.5, 0.2 M NaCl, 2 mM EDTA. Fractions from the chromatogram corresponding to high molecular weight DNA were pooled and used in exonuclease activity determinations. The specific activity of the T7-(3H)-DNA was 6 cpm/pmol of phosphorus equivalents. DNA polymerase activity was determined by a modification of the procedure described by Modrich and Richardson (7). The assay mixture (0.15 ml) contained 93 mM Tris-Cl H 7.6, 2.5 mM DTT, 10 mM MgC12, 53 mM NaCl, 0.15 mM dATP, dGTP, dCTP, and(St H)dTTP (2-5 cpm/pmol), 0.3 mg/ml of bovine serum albumin and 0.5 mM heat-denaturated salmon sperm DNA. The incubations were carried out for 30 min at 370C or 2 min at 17oC. One unit of DNA polymerase catalyzes the incorporation of 10 nmol of total nucleotide into an acidinsoluble product in 30 min at 370C (7). Exonuclease activities were assayed in the mixture of 100 mM Tris, pH 7.5, 10 mM MgC12, 2.5 mM DTT and 2.2 nmol of either native or heat-denaturated T7-(3H)-DNA in a final volume of 0.1 ml as described previously (13). The incubation time was 30 min at 37OC. One unit of exonuclease activity 1411
Vol. 122, No. 3, 1984
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE THE
ELECTROTHERMAL
I
ATOMIZATION
Temperature
PROGRAMME
Ramp
OC
time set
Hold
time set
Drying
100
5
20
Drying
200
5
10
Charring
400
6
24
Atomization
2 300
6
5
Cleaning
2 700
1
2
Cooling
20
15
5
Cooling
20
1
0
catalyzes the formation of 10 nmol of total acid-soluble nucleotide in 30 min at 37OC. Thioredoxin was assayed with thioredoxin reductase and NADPH using DTNB as the electron acceptor as described elsewhere (15). The reduction of DTNB was followed at 412 nm. Protein concentration was determined either by reading the absorbance at 280 nm (13) or by amino acid analysis. T7 DNA polymerase or the gene 5 protein samples were lyophilized and hydrolyzed with 6 M HCl - 0.5% phenol for 24 hours at IlOoC in vacua. Amino acid analysis was performed with a Beckman 121 M amino acxanalyzer. For the calculations,the total compositions of the gene 5 protein (16) and thioredoxin (13) were used. To remove loosely bound or contaminating zinc ions, T7 DNA polymerase and the standard metalloenzymes were applied to a Sephadex G-25 column before the zinc analysis. The column was prewashed with 5 mM EDTA (for 24 h) and equilibrated with 40 mM Tris-Cl buffer, pH 7.5. The equilibration took several days before a constant zinc background (5 rig/ml) was reached. In some experiments all the buffers and enzyme assay mixtures were passed through a column of Chelex-100 which had been converted to the magnesium form by washing with 1 M MgC12 (two bed volumes). Zinc was determined by atomic absorption spectrophotometry (AAS) at 213.8 nm (band-pass 0.7 nm) using an instrument (Perkin Elmer 373) equipped with an electrothermal atomization (ETA) unit (HGA-500), an automatic sample injector (AS-40), a printer (PRS-10)anda two-pen recorder (Model 56). A Zn hollow cathode lamp was used together with the deuterium background correction system. The ETA unit was programmed as shown in Table 1. The purging gas was 99.99% pure argon at an internal flow rate of 300 ml/ min (atomization step). This high gasflow combined with a long ramp time atomization was used to reduce the non-specific background signal to an acceptable level. Injections (20 ~1) of zinc standards or samples were made into standard graphite tubes. The areas under the atomization-signal peaks were integrated (11 s) by the instrument and used for evaluating the results after received on the printer. The recorder was used as a processcontroller with one pen recording the deuterium-compensated Zn signal and the other the non-specific background signal. All determinations were made in duplicate with at least two injections from each cup in AS-40. Zinc standards were prepared for each analysis by dilutions of a commercial standard solution with the buffer used in each experiment. All tips and vessels were carefully acid washed. Method of standard additions was used whenever possible. 1412
Vol.
122,
No.
BIOCHEMICAL
3, 1984
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS Preparation 5 protein
and
graphy the
at
pH
of
mM EDTA, The
polymerase
or
exonuclease
(fig.
to
the
gene
5 protein
of of
2A).
G-25
of
zinc
the
bound
A peak
DNA polymerase ty
20,000
of of
zinc
was
peak
DNA polymerase
chromatography,
or was
while
the
separated
by
Sephadex
and
fractions 0.2
M NaCl,
the
the
of
less
to
of
peaks,
thioredoxin.
the
than had
but
zinc
DNA
content
equilibrated
pH protein
2::
was
residual
almost
T7
no
zinc.
DNA
In
zinc
changed
was buffer
followed
the
specific
after
decreased
the
Sephadex
substantial-
10
Effluent
Figure
1.
- Separation column to gene Gene 5 tivity tivity
(ml)
of the subunits of phage T7 DNA polymerase on a Sephadex G-50 at pH 11.5. The first peak corresponds 5 protein (e+) the second to thioredoxin (o------o). protein activity was measured as T7 3NA polymerase acafter complementation with thioredoxin; thioredoxin acwas followed as a reduction of DTNB (see Methods). of
1413
T7
activi-
1
5
two
order
polymerase
The
had
and
metal-free
activity.
content
polymerase
ly.
0
7.5,
a single-stranded
of
with
significantly
total
50X
purified
polymerase
not
protein
50 mM Tris-Cl,
- T7
for
observed,
two
chromato-
mg protein.
aliquot G-25
gene
G-50
second
and
polymerase
analyzed
the
about
activity
DNA
in
against
exonuclease per
DNA polymerase,
resulted
contained
an
Sephadex
T7
preparation
T7
zinc,
protein T7
in
of
5 protein
units
were
a column
gene
5 protein
double-stranded
loosely to
the
subunits
chromatography
1 mM DTT,
zinc-metallonezymes
applied
were The
gene
activity
remove
(fig.
I). to
Determination
- The
thioredoxin,
5'r0 glycerol,
recovered.
known
5 protein
corresponding dialysis
0.1
gene
E. coli -___
high
first
After
of
Vol. 122, No. 3, 1984
BIOCHEMICAL AND EGOPHYSICAL RESEARCH COMMUNICATIONS
r
L G
L
L)
n
0
”
L
Effluent
Figure
2.
of
symmetrical
genase
was
of
protein
to
have
the
of
standard in
Effect double-stranded creasing
T7
DNA of
carbonic
peak
ratio
the
found
human
found
zinc-protein and
*
0
ImU
- Chromatography of T7 DNA polymerase, preparation III (A) and human carbonic anhydrase (B) on a column of Sephadex G-25. The enzymes were applied to the column (0.8 x 20 cm) in 40 mM TrisCl buffer, pH 7.5. Fractions of 400 ~1 were collected and analyzed for the protein concentration ( M), the content of zinc (A---A) and DNA polymerase activity (M).
A sample one
b
.
the
anhydrase and
zinc
expected
three
applied (fig. zinc
2B).
content.
different
the
same
column
showed
Also
alcohol
Table
II
summarizes
T7
DNA
preparations No
zinc-metalloenzymes.
to
of
stoichiometric
dehydro-
amounts
the
polymerase of
Zn were
polymerase. zinc
and
EDTA
exonuclease concentrations
on
enzyme
activities of
zinc
activities were
to
the
assay
TABLE Zn ANALYSIS
OF 77 DNA POLYMERASE T7 DNA polymerase
Enzyme
inhibited mixtures
T7
DNA by (fig.
polymerase
addition 3A
AND THE STANDARD activity
B).
Zn content
dehydrogenase
3.9
Carbonic
anhydrase
1.1 prep.
I
10,000
0.27
prep.
II
13,000
0.17
prep.
III
10,600
0.35
1414
and
in-
ENZYMES
Alcohol
T7 DNA polymerase
of
II
g atoms/mole
units/mg
and
enzyme
The
Vol.
122,
No.
BIOCHEMICAL
3, 1984
AND
0 ZnCI,
Figure
same
3.
was gene
further
addition
hibition
was
veal of
any up
to Since
low
zinc
zinc-free mined
obtained
for
5 protein of reversed
effect
on
L
I
1
2
orEOTA
COMMUNICATIONS
Imtl,
the
single-stranded
(fig.
3'2).
The
magnesium
ions
to
by
addition
polymerase
of or
exonuclease
activities
were
the
assay
mixtures.
EDTA
(fig.
3).
exonuclease
activity
not
recovered
EDTA
the
itself in
the
by
However,
activities
of
did
in-
not
re-
concentration
2 mM. T7
DNA
polymerase
contamination enzyme.
with
RESEARCH
- The effect of ZnCl? and EDTA on DNA polymerase (A) and doublestranded DNA exonuclease (B) activities of T7 DNA polymerase and single-stranded DNA exonuclease activity of gene 5 protein (C). All three activities were measured in the presence of indicated amounts of EDTA (N) or ZnCl 2d (A+). The reactions of Zninhibited enzyme activities by a ditlon of EDTA are also shown (o----o): The enzyme was then preincubated for 10 min at 4'C in the assay mixtures containing 0.2 mM ZnC12 prior to addition of EDTA followed by the incubation for 30 min at 37OC. T7 DNA polymerase concentrations were 0.85 nM (A) and 1.28 nM (B), respectively. The concentration of gene 5 protein was 1.25 nM (C).
result
isolated
BIOPHYSICAL
higher
normally
present To
exclude
concentration
in this
is the
assay
possibility, of
T7
DNA
1415
assayed
at
mixture enzyme polymerase.
nM
concentrations,
could
a
reactivate
a
activity
was
By
a short
using
deterin-
Vol.
122,
No.
3, 1984
BIOCHEMICAL
AND
ZnCL,or
Figure
4.
cubation was
and
possible
100 fig.
to
without in
the
4,
decreasing
enzyme
EOTA lmMi
hibition
obtained amount
uM of
EDTA,
was
pretreated
form
and was
by
T7
DNA by
adding
incubation The
passage less by
of
the
polymerase.
unaffected
first
of
contained
addition
by
of
temperature
0.2
activity
inhibited
the
assay
magnesium
strongly
molar
COMMUNICATIONS
- The effect of ZnCl and EDTA on DNA polymerase activity at enzyme concentrate activity was measured .5 n. The polymerase the presence of EDTA (e--r ), ZnCl (LA) or EDTA and in equimolar amounts (o----o ). The 2 oncentration of T7 DNA meraie in the assay mixture was 0.2 PM and the incubation carried out for 2 min at 170C. The enzyme was first chromatographed through the column of zinc-free Sephadex G-25 and incubation mixture without DTA was treated by Chelex-100 magnesium form to remove Zn St ions.
time
mixture
BIOPHYSICALRESEARCH
additions
zinc.
As
zinc
was
in
uM
of
of
zinc.
Chelex-
As
excess
previous
shown
EDTA,
reversed
in
but
experiments,
partially
it
assay
a column
0.15
the in
17'C
polymerase
through than
to
high in ZnC12 polvwas -
the by
an
in-
equi-
EDTA.
DISCUSSION The
results
Sephadex
G-25
all
addition
of
functional
role
questioned
by
results
for
for Zn
2+
study
show
excess for the
in
results that
replication extending
activities Zn'+,
Zn
phage no
of
the
T7
DNA
enzyme
were
of
EDTA
addition
the
DNA
polymerase
reaction
for
DNA polymerase
I from
T7
essential DNA
generalization
(7,
virus-induced 8), that
metalloenz.ymes. 1416
polymerase
stoichiometric
whereas
the of
the
that
contained
enzyme
demonstrate
quired
this
chromatography,
Furthermore, by
of
shows DNA
amounts
of
strongly showed (1, E. -__
no
effect.
was (5,
A
recently 6).
Our
DNA polymerase
functional
polymerases
Zn.
inhibited
2)
coli
T7 no
after
re-
requirement are
not
Zn-
Vol.
122,
No.
We have 5 protein which
developed
based is
BIOCHEMICAL
3, 1984
used
on in
the
polymerase
enzyme,
the
gene
hibited
by
Zn2+
binding
of
Zn and
the
the
isolate
5 protein
a novel
thioredoxin
simple
stability
(13,
In to
14).
sulfhydryl contain
cases
T7
Similar
RESEARCH
to
DNA
affinity
single-stranded both
BIOPHYSICAL
technique of
immunoadsorbent
5 protein
ions
and
known
ions.
2+
AND
groups essential
isolate
polymerase
the at
chromatography to
the
T7
inhibition on
the sulfhydryl
protein.
pH,
method
be
was caused
Both groups
to holo-
activity may
gene
high
DNA polymerase
exonuclease this
COMMUNICATIONS
inby
the (12,
gene 17).
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. Barbro Sbderman and Mrs. Agneta Slaby is gratefully acknowledged. Ivan Slaby was supported by a long-term EM90 fellowship. This investigation was supported by grants from the Swedish Cancer Society, project 961, the Swedish Medical Research Council, 13X-3529 and the Knut and Alice Wallenberg Foundation. REFERENCES iI . Sorinqate, C.F., Mildvan, A.S., Abramson, R., Engle, J.L. and Loeb, L:A. (1973) J. Biol. Chem. 248, 5987-5993. Mildvan, A.S. and Loeb, L.A. (1979) CRC Critical Rev. Biochem. 6, 2. 219-244. Coleman, J.E. (1983) In: Zinc Enzymes, Ed. T.T. Spiro, John Wiley & 3. Sons, New York, pp. 221-252. Kornberg, A. (1980) DNA replication. W.H. Freeman & Co., San Francisco, 4. pp. l-724. Herrman, M.S. and Behnke, W.D. (1982) 5. Walton,K.E.,Fitzgerald, P.C., Biochem. Biophys. Res. Commun. 108, 1353-1361. 6. Ferrin, L.J., Mildvan, A.S. and Loeb, L.A. (1983) Biochem. Biophys. Res. Commun. 112, 723-728. 7. Modrich, P. and Richardson, C.C. (1975) J. Biol. Chem. 250, 5508-5522. 8. Mark, D.F. and Richardson, C.C. (1976) Proc. Natl. Acad. Sci. USA 73, 780-784. 9. Hori, K., Mark, D.F. and Richardson, C.C. (1979) J. Biol. Chem. 254, 11591-11597. IO. Hori, K., Mark, D.F. and Richardson, C.C. (1979) J. Biol. Chem. 254, 11598-11604. 11. Adler, S. and Modrich, P. (1979) J. Biol. Chem. 254, 11605-11614. 12. Adler, S. and Modrich, P. (1983) J. Biol. Chem. 258, 6956-6962. 13. Nordstrom, B., Randahl, H., Slaby, I. and Holmgren. A. (1981) 3. Biol. Chem. 256, 3113-3117. 14. Randahl, H., Slaby, I. and Holmgren, A. (1982) Eur. J. Biochem. 128, 445-449. 15. Slaby, I. and Holmgren, A. (1975) J. Biol. Chem. 250, 1340-1347. Dunn, J.J. and Studier, F.W. (1983) J. Mol. Biol. 166, 477-535. 167: Randahl, H. and Holmgren, A. (1984) J. Biol. Chem. submitted for publication.
1417