- Email: [email protected]
Inhibition of DNA polymerase by histones
BIOCHEMICAL
Vol. 14, No. 6, 1964
INRIBITION Lawrence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF DNA POLYMFRASE BY HISTONES
R. Gurley',
3. Logan
Irvin,
and David
J. Holbrook
Department of Biochemistry, School of Medicine University of North Carolina, Chapel Hill
Received
16, 1963
December
Previous
work
in this
laboratory
of DNA, RNA, and histones et al., upon
1962),
(Evans,
incorporation
ascites
preparing
ratio
of histones
for
before
and (b)
et al.,
'1963)
According results
2:l) (1962),
have reported
that
RNA synthesis
through
which Allfrey,
histones
may play
inhibition
of Ehrlich
an important
in
the rising
that
histone
syn-
RNA synthesis
and,
nucleic
(1963),
that
synthesis
at intermediate
immediately
--et al.
fact
inhibits
of both
acid
hypothesis,
DNA synthesis
occur
histones
has led us to suggest
from the
synthesis
acids
of nucleic
(a)
(Holbrook,
of added
nucleic
to this
DNA synthesis:
inhibits
(approaching
Huang and Bonner
into
stimulates
liver
and on the effects
precursors
to DNA, which
of synthesis
of regenerating
the regulation
mitosis.
or indirectly,
the ratio;
in
nuclei
1962)
(Irvin,
a role
commences
directly
ratios
cells
may play
cells
thesis
et al.,
of labeled
carcinoma
histones
in cell
on the time-course
acids
preceding Barr role
of the DNA-dependent
values
at the high mitosis.
and Butler
(1963)
in regulation RNA polymerase.
lAided by grants from the American Cancer Society National Institute of General Medical Sciences,
(E-24E) and the N.I.H. (GM-08318-03).
'Predoctoral
(5 Fl
Fellow
of the Public
Health 527
Service
GM 12,519).
of
of
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The present communication is concerned with a study of the effects histones upon the activity
of
of a DNApolymerase system from regenerating
liver. Methods.
The effect
of histones on DNAsynthesis was measured by use
of an enzyme preparation 1963).
rat liver
Two types of experiments were conducted:
the effect w
from regenerating
Type
(Behki and Schneider, 1, an assay for
of histones on thymidine kinase and deoxyribonucleotide
an assay for the effect
of histones on DNApolymerase.
kinases.
Each of
these assays involved two successive incubations or stages which were performed by mixing 0.45 ml of enzyme and 0.45 ml of incubation medium3 containing H3-thymidine (Behki and Schneider, 1963). was added either
0.05 ml aqueous calf
thymus histone solution
type 1 kinase assay, or 0.05 ml water in the assay.
To this mixture
type
in the
2 DNApolymerase
The mixture was incubated 30 minutes at 380 to generate deoxy-
ribonucleoside
triphosphates.
The reaction was stopped by heating
1 minute at 100' followed by cooling on ice. was removed by centrifugation. 0.1 ml calf
or 0.05 ml histone solution
protein
The supernatant (0.45 ml) was mixed with
thymus DNAprimer solution
this mixture was added either
The precipitated
(1 mg DNA/ml 0.001 M NaCl).
To
0.05 ml water in the type 1 kinase assay in the
type
2 DNApolymerase assay; 0.40 ml
of the enzyme was added and the mixture incubated 30 minutes at 38' to synthesize DNA. The reaction was stopped by adding 0.2 ml 6 N NaOH. The DNAwas then isolated from the mixture
(Orlov and Orlova,
1962),
30.45 ml of incubation mediumcontained 100 voles each of 5'-deoxycytidylic acid, 5'-deoxyadenylic acid, 5'-deoxyguanylic acid; 10 moles adenosine triphosphate, 12 poles potassium 3-phosphoglycerate, 10 poles MgC12, 80 poles tris(hydroxymethyl)aminomethane buffer, pH 8.0, 120 voles (methyl-H3)thymidine, 10 mc/mmole. 528
Vol. 14, No. 6, 1964
washed
BIOCHEMICAL
once with
twice
with
with
ethanol
Samples
taken
using
specific
Bray's
activity
(McAllister,
(Johns
and Butler,
1962).
(McAllister, calf
stages tone
In all while
fractions
DNA,
varying
(Ui,
directly
fractions
1960).
The
glands histone
frac-
1957);
and a
from calf
are
The sodium
thymus
electrophoretically salt
of highly
from Sigma Chemical
the concentration
in terms
l),
35% inhibition
of DNA synthesis occurred
the DNA polymerase
present
histones
2) (Figure
assay
were only
of 4.0.
caused
only
of 4.0
(Figure
were for
of varying added
2).
529
at the two
effects
of his-
histone:DNA
ratios.
of 1.5 or less. of 2.0, histones
inhibition
and loo"/, added
to
of DNA synthesis
of 2.0 or less Lysine-rich
assay
of DNAsynthesis
ratios
Arginine-rich
ratios
the
inhibition
at a ratio
a slight
added
to the DNA polymerase
at histone/DNA
20%) at histone/DNA at a ratio
data
a slight
occurred
at a ratio
of DNA was held
of histones
The figures
on DNA synthesis
type
thymus
histones
1, was purchased
concentrations
15%) was observed
inhibition
the
Scintillation
(Bray,
from calf
1963).
type
(approximately
(approximately
in 0.05
and for
and lysine-rich
These
the experiments,
When unfractionated
inhibition
salts,
as DNA primer.
of incubation.
(experiment
Liquid
was prepared
et al.,
thymus
Company and used
constant
Tricarb
from the unfractionated fraction
Results.
1956)
mixture
prepared
arginine-rich
histone
polymerized
soluble
(Burton,
scintillation
were
1963);
prepared
heterogeneous
lipid,
The DNA was dissolved
in a Packard
lysine-rich
glands
the TCA.
liquid
to remove
to remove water
DNA analysis
histones
et al.,
were
(TCA)
(3:l)
of the DNA in cpm/mg was calculated.
Unfractionated
very
for
of radioactivity
Spectrometer
mixture
acid
to remove
were
determination
tions
alcohol:ether
5% trichloroacetic
and twice NaOH.
40-50'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and only
histones
were
68% more
N
Vol. 14, No. 6, 1964
inhibitory
BIOCHEMICAL
in the DNA polymerase
100% inhibition (Figure more
at ratios
3). than
Very the
of 0.5,
lysine-rich
lysine-rich
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
assay 1.0,
histones histones
and caused 1.5,
2.0,
12%, 35%, 47%, 80% and and 4.0,
inhibited
DNA synthesis
at the higher
ratios
slightly
(Figure
(FIG.
-I
I Hislone /DNA
respectively
I
tibtio
2)
2 Hi&w/DNA
scm
4).
(FIG.
3
Ratio
4)
of histone fractions on DNA synthesis. Figures 1, 2, 3, and 4: The effect Each point in the figures is the average of two determinations. The specific activity of the isolated DNA was corrected for small nonspecific radioactive contamination by subtracting c.p.m. for a control sample in which DNA primer was present but DNA synthesis was prevented by addition of NaOH to the enzyme before incubation.
When various (experiment was observed may result
type
fractions l),
(Figures from
only
of histones a small,
or no,
1, 2, 3 and 4).
incomplete
removal
were
to kinase
inhibition
The small,
of histones 530
added
assay
of DNA synthesis apparent
by the
inhibitions
1 minute
heat
4
Vol. 14, No. 6, 1964
BIOCHEMICAL
treatment between the first
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and second incubation leaving trace amounts
of active histones in the supernatant during the DNApolymerase incubation.
these small, apparent effects
Therefore,
assay possibly can be interpreted
of histone on the kinase
as residual effects
on the polymerase
rather than on the kinases. Discussion.
Huang and Bonner (1962) have suggested that histones may
control RNAsynthesis by inhibiting
BNA polymerase.
They have shown,
&I vitro,
that at a histone/DNA ratio
of 1.0, ENA synthesis is completely
inhibited
in a system isolated from pea seedling nuclei.
presented in this report show that at this ratio, rich histone fraction a lysine-rich
causes only a 21%inhibition
histone fraction
At a histone/DNA ratio
inhibits
-in vitro,
an arginine-
of DNAsynthesis,
inhibits
and
DNA synthesis only about 35%.
of 2.0, however, DNA synthesis is still
only moderately by the arginine-rich histone fraction
The data
histone fraction,
affected
but the lysine-rich
DNAsynthesis by 80%. These data lend somesup-
port to the previous experiments -in vivo
(Irvin,
e& &.,
1963) from the
results of which it was proposed that DNAsynthesis is stopped when high histone/DNA ratios
are attained in nuclei preparing for mitosis while RNA
synthesis declines at lower ratios.
Preliminary
vations of E. L. Cooler, R. F. Fisher, liver
indicates
before mitosis. inhibitor possibility
work (unpublished obser-
and J. L. Irvin)
that a histone/DNA ratio
of about 2 is reached shortly
The fact that the lysine-rich
fraction
of DNApolymerase than the arginine-rich that the lysine-rich
cessation of DNAsynthesis prior
fraction
is a stronger
fraction
is particularly
to mitosis.
531
on regenerating
suggests the involved
in
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References 1. Allfrey, V. G., Littan, V. C., and Mirsky, A. E., Proc. Natl. Acad. Sci. U. S ., 49. 414 (1963). 2. Barr, G. C., and Butler, J. A. V., Nature, 199, 1170 (1963). W. C., Biochim. Biophys. Acta, a 34 3. Behki, R. M., and Schneider, (1963). 4. Bray, G. A., Analyt. Biochem., L 279 (1960). 5. Burton, K., Biochem. J., 62, 315 (1956). 6. Evans, J. H., Holbrook, D. J., Jr., and Irvin, J. L., Rxptl. Cell Res ., 28- 126 (1962). 7. Holbrook, D. J., Jr., Evans, J. H., and Irvin, J. L., Exptl. Cell Res., 28, 120 (1962). 8, Huang, R. C. and Bonner, J., Proc. Natl. Acad. Sci. U. S., 48, 1216 (1962). 9. Irvin, J. L., Holbrook, D. J., Jr., Evans, J. H., McAllister, H. C., and Stiles, E. P., Rxptl. Cell Res., Suppl. 2, 359 (1963). J. A. V., Biochem. J., 82, 15 (1962). 10. Johns, E. W., and Butler, 11. McAllister, PI. C., Jr., Nan, Y. C., and Irvin, J. L., Analyt. Biochem., zs 321 (1963). 12. Orlov, A. S. and Orlova, E. I., Biochemistry, 26, 722 (1962), the English translation of Biokhimiya 26, 834 (1961). Biophys. Acta, 25, 493 (1957). 13. Ui, N., Biochim.
532
Vol. 14, No. 6, 1964
INRIBITION Lawrence
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF DNA POLYMFRASE BY HISTONES
R. Gurley',
3. Logan
Irvin,
and David
J. Holbrook
Department of Biochemistry, School of Medicine University of North Carolina, Chapel Hill
Received
16, 1963
December
Previous
work
in this
laboratory
of DNA, RNA, and histones et al., upon
1962),
(Evans,
incorporation
ascites
preparing
ratio
of histones
for
before
and (b)
et al.,
'1963)
According results
2:l) (1962),
have reported
that
RNA synthesis
through
which Allfrey,
histones
may play
inhibition
of Ehrlich
an important
in
the rising
that
histone
syn-
RNA synthesis
and,
nucleic
(1963),
that
synthesis
at intermediate
immediately
--et al.
fact
inhibits
of both
acid
hypothesis,
DNA synthesis
occur
histones
has led us to suggest
from the
synthesis
acids
of nucleic
(a)
(Holbrook,
of added
nucleic
to this
DNA synthesis:
inhibits
(approaching
Huang and Bonner
into
stimulates
liver
and on the effects
precursors
to DNA, which
of synthesis
of regenerating
the regulation
mitosis.
or indirectly,
the ratio;
in
nuclei
1962)
(Irvin,
a role
commences
directly
ratios
cells
may play
cells
thesis
et al.,
of labeled
carcinoma
histones
in cell
on the time-course
acids
preceding Barr role
of the DNA-dependent
values
at the high mitosis.
and Butler
(1963)
in regulation RNA polymerase.
lAided by grants from the American Cancer Society National Institute of General Medical Sciences,
(E-24E) and the N.I.H. (GM-08318-03).
'Predoctoral
(5 Fl
Fellow
of the Public
Health 527
Service
GM 12,519).
of
of
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The present communication is concerned with a study of the effects histones upon the activity
of
of a DNApolymerase system from regenerating
liver. Methods.
The effect
of histones on DNAsynthesis was measured by use
of an enzyme preparation 1963).
rat liver
Two types of experiments were conducted:
the effect w
from regenerating
Type
(Behki and Schneider, 1, an assay for
of histones on thymidine kinase and deoxyribonucleotide
an assay for the effect
of histones on DNApolymerase.
kinases.
Each of
these assays involved two successive incubations or stages which were performed by mixing 0.45 ml of enzyme and 0.45 ml of incubation medium3 containing H3-thymidine (Behki and Schneider, 1963). was added either
0.05 ml aqueous calf
thymus histone solution
type 1 kinase assay, or 0.05 ml water in the assay.
To this mixture
type
in the
2 DNApolymerase
The mixture was incubated 30 minutes at 380 to generate deoxy-
ribonucleoside
triphosphates.
The reaction was stopped by heating
1 minute at 100' followed by cooling on ice. was removed by centrifugation. 0.1 ml calf
or 0.05 ml histone solution
protein
The supernatant (0.45 ml) was mixed with
thymus DNAprimer solution
this mixture was added either
The precipitated
(1 mg DNA/ml 0.001 M NaCl).
To
0.05 ml water in the type 1 kinase assay in the
type
2 DNApolymerase assay; 0.40 ml
of the enzyme was added and the mixture incubated 30 minutes at 38' to synthesize DNA. The reaction was stopped by adding 0.2 ml 6 N NaOH. The DNAwas then isolated from the mixture
(Orlov and Orlova,
1962),
30.45 ml of incubation mediumcontained 100 voles each of 5'-deoxycytidylic acid, 5'-deoxyadenylic acid, 5'-deoxyguanylic acid; 10 moles adenosine triphosphate, 12 poles potassium 3-phosphoglycerate, 10 poles MgC12, 80 poles tris(hydroxymethyl)aminomethane buffer, pH 8.0, 120 voles (methyl-H3)thymidine, 10 mc/mmole. 528
Vol. 14, No. 6, 1964
washed
BIOCHEMICAL
once with
twice
with
with
ethanol
Samples
taken
using
specific
Bray's
activity
(McAllister,
(Johns
and Butler,
1962).
(McAllister, calf
stages tone
In all while
fractions
DNA,
varying
(Ui,
directly
fractions
1960).
The
glands histone
frac-
1957);
and a
from calf
are
The sodium
thymus
electrophoretically salt
of highly
from Sigma Chemical
the concentration
in terms
l),
35% inhibition
of DNA synthesis occurred
the DNA polymerase
present
histones
2) (Figure
assay
were only
of 4.0.
caused
only
of 4.0
(Figure
were for
of varying added
2).
529
at the two
effects
of his-
histone:DNA
ratios.
of 1.5 or less. of 2.0, histones
inhibition
and loo"/, added
to
of DNA synthesis
of 2.0 or less Lysine-rich
assay
of DNAsynthesis
ratios
Arginine-rich
ratios
the
inhibition
at a ratio
a slight
added
to the DNA polymerase
at histone/DNA
20%) at histone/DNA at a ratio
data
a slight
occurred
at a ratio
of DNA was held
of histones
The figures
on DNA synthesis
type
thymus
histones
1, was purchased
concentrations
15%) was observed
inhibition
the
Scintillation
(Bray,
from calf
1963).
type
(approximately
(approximately
in 0.05
and for
and lysine-rich
These
the experiments,
When unfractionated
inhibition
salts,
as DNA primer.
of incubation.
(experiment
Liquid
was prepared
et al.,
thymus
Company and used
constant
Tricarb
from the unfractionated fraction
Results.
1956)
mixture
prepared
arginine-rich
histone
polymerized
soluble
(Burton,
scintillation
were
1963);
prepared
heterogeneous
lipid,
The DNA was dissolved
in a Packard
lysine-rich
glands
the TCA.
liquid
to remove
to remove water
DNA analysis
histones
et al.,
were
(TCA)
(3:l)
of the DNA in cpm/mg was calculated.
Unfractionated
very
for
of radioactivity
Spectrometer
mixture
acid
to remove
were
determination
tions
alcohol:ether
5% trichloroacetic
and twice NaOH.
40-50'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and only
histones
were
68% more
N
Vol. 14, No. 6, 1964
inhibitory
BIOCHEMICAL
in the DNA polymerase
100% inhibition (Figure more
at ratios
3). than
Very the
of 0.5,
lysine-rich
lysine-rich
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
assay 1.0,
histones histones
and caused 1.5,
2.0,
12%, 35%, 47%, 80% and and 4.0,
inhibited
DNA synthesis
at the higher
ratios
slightly
(Figure
(FIG.
-I
I Hislone /DNA
respectively
I
tibtio
2)
2 Hi&w/DNA
scm
4).
(FIG.
3
Ratio
4)
of histone fractions on DNA synthesis. Figures 1, 2, 3, and 4: The effect Each point in the figures is the average of two determinations. The specific activity of the isolated DNA was corrected for small nonspecific radioactive contamination by subtracting c.p.m. for a control sample in which DNA primer was present but DNA synthesis was prevented by addition of NaOH to the enzyme before incubation.
When various (experiment was observed may result
type
fractions l),
(Figures from
only
of histones a small,
or no,
1, 2, 3 and 4).
incomplete
removal
were
to kinase
inhibition
The small,
of histones 530
added
assay
of DNA synthesis apparent
by the
inhibitions
1 minute
heat
4
Vol. 14, No. 6, 1964
BIOCHEMICAL
treatment between the first
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and second incubation leaving trace amounts
of active histones in the supernatant during the DNApolymerase incubation.
these small, apparent effects
Therefore,
assay possibly can be interpreted
of histone on the kinase
as residual effects
on the polymerase
rather than on the kinases. Discussion.
Huang and Bonner (1962) have suggested that histones may
control RNAsynthesis by inhibiting
BNA polymerase.
They have shown,
&I vitro,
that at a histone/DNA ratio
of 1.0, ENA synthesis is completely
inhibited
in a system isolated from pea seedling nuclei.
presented in this report show that at this ratio, rich histone fraction a lysine-rich
causes only a 21%inhibition
histone fraction
At a histone/DNA ratio
inhibits
-in vitro,
an arginine-
of DNAsynthesis,
inhibits
and
DNA synthesis only about 35%.
of 2.0, however, DNA synthesis is still
only moderately by the arginine-rich histone fraction
The data
histone fraction,
affected
but the lysine-rich
DNAsynthesis by 80%. These data lend somesup-
port to the previous experiments -in vivo
(Irvin,
e& &.,
1963) from the
results of which it was proposed that DNAsynthesis is stopped when high histone/DNA ratios
are attained in nuclei preparing for mitosis while RNA
synthesis declines at lower ratios.
Preliminary
vations of E. L. Cooler, R. F. Fisher, liver
indicates
before mitosis. inhibitor possibility
work (unpublished obser-
and J. L. Irvin)
that a histone/DNA ratio
of about 2 is reached shortly
The fact that the lysine-rich
fraction
of DNApolymerase than the arginine-rich that the lysine-rich
cessation of DNAsynthesis prior
fraction
is a stronger
fraction
is particularly
to mitosis.
531
on regenerating
suggests the involved
in
Vol. 14, No. 6, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
References 1. Allfrey, V. G., Littan, V. C., and Mirsky, A. E., Proc. Natl. Acad. Sci. U. S ., 49. 414 (1963). 2. Barr, G. C., and Butler, J. A. V., Nature, 199, 1170 (1963). W. C., Biochim. Biophys. Acta, a 34 3. Behki, R. M., and Schneider, (1963). 4. Bray, G. A., Analyt. Biochem., L 279 (1960). 5. Burton, K., Biochem. J., 62, 315 (1956). 6. Evans, J. H., Holbrook, D. J., Jr., and Irvin, J. L., Rxptl. Cell Res ., 28- 126 (1962). 7. Holbrook, D. J., Jr., Evans, J. H., and Irvin, J. L., Exptl. Cell Res., 28, 120 (1962). 8, Huang, R. C. and Bonner, J., Proc. Natl. Acad. Sci. U. S., 48, 1216 (1962). 9. Irvin, J. L., Holbrook, D. J., Jr., Evans, J. H., McAllister, H. C., and Stiles, E. P., Rxptl. Cell Res., Suppl. 2, 359 (1963). J. A. V., Biochem. J., 82, 15 (1962). 10. Johns, E. W., and Butler, 11. McAllister, PI. C., Jr., Nan, Y. C., and Irvin, J. L., Analyt. Biochem., zs 321 (1963). 12. Orlov, A. S. and Orlova, E. I., Biochemistry, 26, 722 (1962), the English translation of Biokhimiya 26, 834 (1961). Biophys. Acta, 25, 493 (1957). 13. Ui, N., Biochim.
532