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Temperature dependent permeability changes of Bacillus subtilis and incorporation of nucleotides into DNA
BIOCHEMICAL
Vol. 55, No. 3,1973
TEMPERATURE
AND BlOPHYSlCAL
DEPENDENT PERMEABILITY AND INCORPORATION
RESEARCH COMMlJNlCATlONS
CHANGES OF BACILLUS
OF NUCLEOTIDES
SUBTILIS
INTO DNA
R. C. HENNEBERRY and ERNST FREESE
National
Laboratory of Molecular Biology Institute of Neurological Diseases National Institutes of Health Bethesda, Maryland 20014
Received
September
and Stroke
24,1973 SUMMARY
Cells of B. subtilis exposed to temperatures between 0 and 5 C are permeable to small molecules not normally able to pass through the cell envelope. As a result, deoxyribonucleotide triphosphates (dXTPs) are incorporated into DNA if the reaction mixture contains all four dXTPs. Since this incorporation is insensitive to 6-(p-hydroxyphenylazo)-uracil and is not observed in DNA Polymerase I mutants, we conclude it reflects DNA repair rather than the DNA replication which can be observed in cells permeabilized with toluene.
INTRODUCTION The sudden
chilling
shock")
causes
the
viability
in
a variety
shock
was generally
bacteria
(S),
the
release
slow
We report leakage
here of cell
ribonucleotide changes ment
release
of microorganisms thought
Smeaton
and Elliott
(11)
cold
material
ineffective.
Copynkht 0 1973 by Academic Press, inc. AN rights of reproduction in any form reserved.
and loss
Although
of
cold
on Gram positive
reported
that
it
caused
of Bacillus
subtilis.
treatment
of -B. subtilis causes the and permits the incorporation of deoxy-
at temperatures
8 C is
(5).
inhibitor
to 0 C ("cold
material
to have no effect
of a ribonuclease that
suspension
of UV absorbing
triphosphates
occur
above
of a bacterial
(dXTPs) between Mutant 788
into
DNA.
These
permeability
0 and 5 C, whereas and inhibitor
treat-
studies
Vol. 55, No. 3,1973
show that
the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dXTP incorporation
than the DNA replication toluene
reflects
observed
in cells
rather
permeabilized
with
(7).
MATERIALS
tics
DNA repair
AND METHODS
The strains of B. subtilis employed and their characterisare shown in Table 1. Cells were grown in phosphate-
TABLE I g. Strain
Source
60015
E. Nester
60437
B. Strauss
60439
B. Strauss
61476
N. Brown
61304
J.
Gross
(SB26)
subtilis
Growth
Strains requirements
Relevant
characteristics
trp,
met
Standard
(JBl-49-40)
trp,
thy
(JBl-49-14)
trp,
thy
Sensitive
(NB841)
trp,
thy
DNA Polymerase
(ts134)
trp,
thy
Sensitive
strain to UV and MMS
to W but
not
I deficient
ts DNA B (initiation)
buffered nutrient sporulation medium (NSMP, ref. 6) supplemented with met (10 ug/ml), thy (5 ug/ml), and trp (25 ug/ml) where shaker to an absorbneeded at 37 C in a reciprocating waterbath They were centrifuged at room ance at 600 nm (A60R) of-1.0. temperature and was ed with an equal volume of room temperature “phosphate buffer” (0.1 M K2HPO -NaH2P04, pH 7.4). Cells were phosphate normally cold shocked by suspen 1,lng them in ice-cold buffer and incubating the suspension for 5 minutes on ice. When temperatures other than 0 C were used the phosphate buffer was equilibrated at,the test temperature and the cells were incubated To measure the release of UV absorbing at this test temperature. material the cell suspension was filtered through a membrane filter (0.45 u pore size, Millipore) and the A For measurement of [‘8~TF~di%$!p”,f.the filtrate was determined. tion, cells treated for 5 minutes at 0 C were transferred to 37 C and incubated 15 minutes; 6 x lo* of these cells were added to a reaction mixture (600 uliter final volume) containing K-PO (1.3 mM) ; ATP (2 mM) ; and dATP, dCT6, dGTP (70 mM, pH 7.4); MtSO New England (each 33 PM) and [ H]#TP (33 PM, 0.25 uCi/nmole, Nuclear). 100 uliter samples were added to 2 ml ice-cold trichloroacetic acid (TCA, 10%) containing 0.1 M Na4-pyrophosphate. were collected on After 15 minutes or more on ice , precipitates membrane filters that had been soaked in 5% TCA, washed twice with 15 ml cold 5% TCA, dried and counted in a Packard Tri-Carb spectrometer in 10 ml toluene containing PI ScintillatorWsearch Products International).
MMS
BIOCHEMICAL
Vol. 55, No. 3,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Cells times
of B.
at room
absorbed 3,and
assume
between sibly,
consists
when cells
phenomenon
transition
temperature
When the
cold
37 C and then and the
added
four
TCA precipitable
DNA but
of
cannot
-B. subtilis
they
Thymine,
the
indicating
that
some breakdown
for containing
Without
TTP is
a direct (10);
the
cold
790
are
on incorshown in
to dilute
incorporated. which
in
molecules
triphosphate
AMP, or ADP; CTP was somewhat used
of
small
omissions
mixture
by adenosine,
assay
of
envelope.
nucleotide
by ATP,
shock,
shocking to
and/or
is
into
precursor
TMP, and TDP failed
product
Mg++,
(TTP)
cold
cells
cell
reaction
the
15 minutes
13HlTTP
was stimulated
incorporation
phase (4).
mixture
additions
Pos-
temperatures.
incorporation
The TTP
to temperatures
incubated
the
complete
thymidine,
of UV absorbing
2).
through
10
weight
lipids
permeabilizes pass
100 C for
thermotropic
cells
about
low molecular
incorporated
(Figure
of various
of TTP in
than
to a reaction
of
of
release
were
vegetative
cannot
The effects
TTP isotope,
cells
TTP was poor.
apparently
3.
membrane
dXTPs,
enter
normally
poration
of the
same
characterization
at higher to the
material
incorporation
the
to
the
4
material
ratio
heating
about in
The released
of
relates
shocked
treated
were exposed
0 and 5 C and decreased this
cells
further
1 shows that
was greatest
0 C released
2).
after
it
at
an A260/A2So
attempted
Figure
material
rather
not
but
metabolites.
Table
(Table
at 260 nm,had
We have material
that
than
showed no hyperchromicity
this
ATP,
shocked
material
temperature
maximally
minutes.
at
cold
W absorbirrg
more
manner
subtilis
could
itself TTP
not
stimulatory
these
the
studies
be
replaced (Table measures
3).
1.050
0.935
1.088
1
2
3
o
2.438
1.974
2.230
material
from
0.704
0.522
0.519
cold
3.5
3.8
4.3
(OC/23C)
Absorbance
shocked
0.970
0.657
0.745
o-
0.213
0.168
0.150
4.5
3.9
5.0
(OC/23C)
of B. subtilis*
of Filtrates
cells
*Cells from 50 ml culture of B. subtilis strain 60015 were centrifuged and washed at room temperature. The pellet was suspended in 2 ml phosphate buffer at either 0 C or 23 C and after 5 minutes cell-free filtrates were prepared as described in Materials and Methods.
A600 of Culture
of W absorbing
Experiment No.
Release
TABLE 2
BIOCHEMICAL
Vol. 55, No. 3,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60 -
“‘\< \ ‘0
\
\
.
.
\ \ ‘.
40 0
0
10
20
Temperature
(‘C)
filtrates from cells of Figure 1. Absorbance (A260 ) of cell-free B. subtilis strain 60015 cold shocked at the indicated temperature Tsee Materials and Methods).
extracellular However,
as well 80% of
the
as intracellular incorporated
DNA polymerase TTP pelleted
low speed
centrifugation
(3,000
the
in
was rendered
label
DNase
(200
pellet
with
88% of the that
most
intracellular
this
pg/ml,
pellet
20 minutes,
lysozyme tritium of
the
(100 vg/ml, became
8 minutes);
After
than
on 1% of
by pancreatic of
the
37 C) and DNaSe,
These in cold
cells
treatment
30 minutes,
TCA-soluble.
the less
TCA-soluble
37 C).
TTP incorporation
results
shocked
indicate cells
reflects
DNA synthesis.
When a temperature ref.
x g,
with
activity.
8) was shifted
and TTP incorporation
sensitive to
45 C for was then
DNA initiation 45 minutes measured
792
mutant
prior at
to cold
45 C, the
(61304, shock,
same amount
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
50-
Figure 2. g. subtilis
of 3)
[%]TTP strain
incorporated into TCA precipitable 60015 (see Materials and Methods).
incorporation Figure
l
Moreover,
was observed 3 again
as in
shows the
TTP incorporation
inhibitor
in
61304
These
results
cells
reflected
To verify cold
shocked
cells
60439,
(MfG),
incorporated
contrast,
of
sensitive
strain
in g.
subtilis
TTP incorporation
several
repair
to W but
to methyl
TTP at least
as well
60437,
is
which
793
is a (9).
cold
shocked
synthesis.
was examined
deficient not
which
in
replication
by ATP.
to reduced
31,
than
as the
sensitive
by
(Figure
reaction
Figure
TTP incorporation rather
conclusion,
Strain
In
that
DNA repair
this
of the
DNA replication
suggested
strain
was insensitive
(HPUra,
of
standard
stimulation
6-(p-hydroxyphenylazo)-uracil specific
the
material
mutants
in (Figure
methane
sulfonate
standard
strain.
to both
W
4).
and MMS,
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
RESEARCH, COMMUNICATIONS
TABLE 3
Contents
of
Complete II
Reaction
107
II
+ thymidine*
110
If
+ TMP*
83
1,
+ TDP*
80
II
+ TTP*
6
11
- MgS04
11
11
- ATP
15
II
- ATP + adenosine/AMP/ADP**
17-33
11
- ATP + CTP**
55
**2
UM
the
lower
10% of
the
Strauss,
(data
not
levels
as well
as the
absorbing
All
conclusion
incorporation
61477,
the
which
has less
on the
strain
than
incorporated
based
has
TTP very
were permeabilized
strain,
which
parental
incorporated
(9),
strains
standard
even
poorly 0.5% of
less
by cold comparison
TTP
shock of W
released. that
reflects
in
the mixture.
cold
shocked
DNA repair
may explain
37 C before
of
61476,
I activity
these
We conclude incorporation
I activity
The mutant
material
The strain
communication)
Polymerase
4).
of TTP.
Polymerase
shown).
normal
mM
personal
(Figure
at
Incorporated
+ thymine"
incorporated
(B.
% i3H]TTP
100
*165
-nly
Mixture
addition
the
rather
necessity of
cells
cold
Incubation
of B. - subtilis than replication.
for shocked
the
15 minute
cells
at 0 C or in
794
to the the
TTP This incubation TTP
presence
of
Vol. 55, No. 3,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIQNS
MINUTES
Figure 3. [%]TTP incorporated cold shocked (1) C) cells of g. and Methods).
p-hydroxymercuribenzoate incorporation
(Figure
to DNA is
a prerequisite
tion
of
repair
been
reported
ATP-dependent
5).
37 C reduced Presumably,
for
by activation in E.
(3).
trations,
which
This
of
could
explain
subsequent
repair
endogenous with
in g. is
material by (see Materials
TTP
nuclease-mediated
permeabilized
enzyme
the
subsequent
deoxyribonuclease
reported
by
at
into TCA precipitable subtilis strain 61304
synthesis.
nucleases
inhibited
the
stimulation
Induc-
has previously
Tris-EDTA
subtilis
also
damage
(1,2).
has recently by high
An been
ATP concen-
of TTP incorporation
ATP. In
repair allowed
contrast deficient incorporation
to cold
shocking,
mutants
(60437 of TTP,
permeabilization and 61476)
which
795
with
was sensitive
of toluene
the (7)
to HPUra
two
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
5
20-
9 a’ 6 E 8 z
Prevncubated
/
;
IO-
/ /
I 15 MINUTES
I 20
MINUTES
Figure 4. L3HlTTP incorporated cold shocked (0 C) cells of the (see Materials and Methods).
and therefore
represented
had been
shocked
about
50% less
treatment. by the This
Possibly, cold
could
fraction replicate
TTP than
shock
(50%) of their
only
that the
the
cells
and the
imply
into TCA precipitable subtilis strain 60015
DNA replication. before
toluene
which about
other
in
treatment
received
cells
DNA. 796
which
the
toluene
were affected
50% permeabilized
such a way that
cells
incorporated
only
by cold
by
material by (see Materials
However,
50% of the
permeabilization cells
30
into TCA precipitable material indicated strain of II. subtilis
Figure 5. [3H]TTP incorporated cold shocked (0 C) cells of g. and Methods).
cold
at 37OC
/
2
I IO
RESEARCH COMMUNICATIONS
by toluene. shock they
affects
a
can no longer
BIOCHEMICAL
Vol. 55, wo. 3,1973
sure
It
is
to
low temperatures,
refrigerated this
should
apparent
that
centrifuge, be considered
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
permeabilization
of B_. subtilis
as may inadvertently can affect in the
several design
occur cellular
by
expo-
in a processes;
of experiments.
REFERENCES 1.
Buttin,
G.,
and Kornberg,
A.
(1966)
J.
Biol.
Chem., 241,
5419-
5427. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11.
Buttin, G., and Wright, M. (1968) Cold Spring Harbor Symp. 33, 259-269. Chestukhin, A. V., Shemykin, M. F., Kalinina, N. A., and Prozorov, A. A. (1972) FEBS Letters 24, 121-125. Esfahani, M., Limbrick, A. R., Knutton, S., Oka, T., and Wakil, J. (1971) Proc. Nat. Acad. Sci. USA 68, 3180-3184. Farrell, J., and Rose, A. H. (1967) in Therzbiology, A. H. Academic Press, New York. Rose, ed., pp. 147-218, Fortnagel, P., and Freese, E. (1968) J. Bacterial. 95, 14311438. Matsushita, T., White, K. P., and Sueoka, N. (1971) Nature New Biol. 232, 111-114. Mendelson, N. H., and Gross, J. D. (1967) J. Bacterial. 2, 1603-1608. Neville, M., and Brown, N. C. (1972) Nature New Biol. 240, 80-82. Rhinehart, K. V., and Copeland, J. C. (1973) Biochim. mphys. Acta 294, l-7. W. H. (1967) Biochem. Biophys. Res. Smeatc J. R., and Elliott, Commun. 26, 75-81.
797
Vol. 55, No. 3,1973
TEMPERATURE
AND BlOPHYSlCAL
DEPENDENT PERMEABILITY AND INCORPORATION
RESEARCH COMMlJNlCATlONS
CHANGES OF BACILLUS
OF NUCLEOTIDES
SUBTILIS
INTO DNA
R. C. HENNEBERRY and ERNST FREESE
National
Laboratory of Molecular Biology Institute of Neurological Diseases National Institutes of Health Bethesda, Maryland 20014
Received
September
and Stroke
24,1973 SUMMARY
Cells of B. subtilis exposed to temperatures between 0 and 5 C are permeable to small molecules not normally able to pass through the cell envelope. As a result, deoxyribonucleotide triphosphates (dXTPs) are incorporated into DNA if the reaction mixture contains all four dXTPs. Since this incorporation is insensitive to 6-(p-hydroxyphenylazo)-uracil and is not observed in DNA Polymerase I mutants, we conclude it reflects DNA repair rather than the DNA replication which can be observed in cells permeabilized with toluene.
INTRODUCTION The sudden
chilling
shock")
causes
the
viability
in
a variety
shock
was generally
bacteria
(S),
the
release
slow
We report leakage
here of cell
ribonucleotide changes ment
release
of microorganisms thought
Smeaton
and Elliott
(11)
cold
material
ineffective.
Copynkht 0 1973 by Academic Press, inc. AN rights of reproduction in any form reserved.
and loss
Although
of
cold
on Gram positive
reported
that
it
caused
of Bacillus
subtilis.
treatment
of -B. subtilis causes the and permits the incorporation of deoxy-
at temperatures
8 C is
(5).
inhibitor
to 0 C ("cold
material
to have no effect
of a ribonuclease that
suspension
of UV absorbing
triphosphates
occur
above
of a bacterial
(dXTPs) between Mutant 788
into
DNA.
These
permeability
0 and 5 C, whereas and inhibitor
treat-
studies
Vol. 55, No. 3,1973
show that
the
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dXTP incorporation
than the DNA replication toluene
reflects
observed
in cells
rather
permeabilized
with
(7).
MATERIALS
tics
DNA repair
AND METHODS
The strains of B. subtilis employed and their characterisare shown in Table 1. Cells were grown in phosphate-
TABLE I g. Strain
Source
60015
E. Nester
60437
B. Strauss
60439
B. Strauss
61476
N. Brown
61304
J.
Gross
(SB26)
subtilis
Growth
Strains requirements
Relevant
characteristics
trp,
met
Standard
(JBl-49-40)
trp,
thy
(JBl-49-14)
trp,
thy
Sensitive
(NB841)
trp,
thy
DNA Polymerase
(ts134)
trp,
thy
Sensitive
strain to UV and MMS
to W but
not
I deficient
ts DNA B (initiation)
buffered nutrient sporulation medium (NSMP, ref. 6) supplemented with met (10 ug/ml), thy (5 ug/ml), and trp (25 ug/ml) where shaker to an absorbneeded at 37 C in a reciprocating waterbath They were centrifuged at room ance at 600 nm (A60R) of-1.0. temperature and was ed with an equal volume of room temperature “phosphate buffer” (0.1 M K2HPO -NaH2P04, pH 7.4). Cells were phosphate normally cold shocked by suspen 1,lng them in ice-cold buffer and incubating the suspension for 5 minutes on ice. When temperatures other than 0 C were used the phosphate buffer was equilibrated at,the test temperature and the cells were incubated To measure the release of UV absorbing at this test temperature. material the cell suspension was filtered through a membrane filter (0.45 u pore size, Millipore) and the A For measurement of [‘8~TF~di%$!p”,f.the filtrate was determined. tion, cells treated for 5 minutes at 0 C were transferred to 37 C and incubated 15 minutes; 6 x lo* of these cells were added to a reaction mixture (600 uliter final volume) containing K-PO (1.3 mM) ; ATP (2 mM) ; and dATP, dCT6, dGTP (70 mM, pH 7.4); MtSO New England (each 33 PM) and [ H]#TP (33 PM, 0.25 uCi/nmole, Nuclear). 100 uliter samples were added to 2 ml ice-cold trichloroacetic acid (TCA, 10%) containing 0.1 M Na4-pyrophosphate. were collected on After 15 minutes or more on ice , precipitates membrane filters that had been soaked in 5% TCA, washed twice with 15 ml cold 5% TCA, dried and counted in a Packard Tri-Carb spectrometer in 10 ml toluene containing PI ScintillatorWsearch Products International).
MMS
BIOCHEMICAL
Vol. 55, No. 3,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION Cells times
of B.
at room
absorbed 3,and
assume
between sibly,
consists
when cells
phenomenon
transition
temperature
When the
cold
37 C and then and the
added
four
TCA precipitable
DNA but
of
cannot
-B. subtilis
they
Thymine,
the
indicating
that
some breakdown
for containing
Without
TTP is
a direct (10);
the
cold
790
are
on incorshown in
to dilute
incorporated. which
in
molecules
triphosphate
AMP, or ADP; CTP was somewhat used
of
small
omissions
mixture
by adenosine,
assay
of
envelope.
nucleotide
by ATP,
shock,
shocking to
and/or
is
into
precursor
TMP, and TDP failed
product
Mg++,
(TTP)
cold
cells
cell
reaction
the
15 minutes
13HlTTP
was stimulated
incorporation
phase (4).
mixture
additions
Pos-
temperatures.
incorporation
The TTP
to temperatures
incubated
the
complete
thymidine,
of UV absorbing
2).
through
10
weight
lipids
permeabilizes pass
100 C for
thermotropic
cells
about
low molecular
incorporated
(Figure
of various
of TTP in
than
to a reaction
of
of
release
were
vegetative
cannot
The effects
TTP isotope,
cells
TTP was poor.
apparently
3.
membrane
dXTPs,
enter
normally
poration
of the
same
characterization
at higher to the
material
incorporation
the
to
the
4
material
ratio
heating
about in
The released
of
relates
shocked
treated
were exposed
0 and 5 C and decreased this
cells
further
1 shows that
was greatest
0 C released
2).
after
it
at
an A260/A2So
attempted
Figure
material
rather
not
but
metabolites.
Table
(Table
at 260 nm,had
We have material
that
than
showed no hyperchromicity
this
ATP,
shocked
material
temperature
maximally
minutes.
at
cold
W absorbirrg
more
manner
subtilis
could
itself TTP
not
stimulatory
these
the
studies
be
replaced (Table measures
3).
1.050
0.935
1.088
1
2
3
o
2.438
1.974
2.230
material
from
0.704
0.522
0.519
cold
3.5
3.8
4.3
(OC/23C)
Absorbance
shocked
0.970
0.657
0.745
o-
0.213
0.168
0.150
4.5
3.9
5.0
(OC/23C)
of B. subtilis*
of Filtrates
cells
*Cells from 50 ml culture of B. subtilis strain 60015 were centrifuged and washed at room temperature. The pellet was suspended in 2 ml phosphate buffer at either 0 C or 23 C and after 5 minutes cell-free filtrates were prepared as described in Materials and Methods.
A600 of Culture
of W absorbing
Experiment No.
Release
TABLE 2
BIOCHEMICAL
Vol. 55, No. 3,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60 -
“‘\< \ ‘0
\
\
.
.
\ \ ‘.
40 0
0
10
20
Temperature
(‘C)
filtrates from cells of Figure 1. Absorbance (A260 ) of cell-free B. subtilis strain 60015 cold shocked at the indicated temperature Tsee Materials and Methods).
extracellular However,
as well 80% of
the
as intracellular incorporated
DNA polymerase TTP pelleted
low speed
centrifugation
(3,000
the
in
was rendered
label
DNase
(200
pellet
with
88% of the that
most
intracellular
this
pg/ml,
pellet
20 minutes,
lysozyme tritium of
the
(100 vg/ml, became
8 minutes);
After
than
on 1% of
by pancreatic of
the
37 C) and DNaSe,
These in cold
cells
treatment
30 minutes,
TCA-soluble.
the less
TCA-soluble
37 C).
TTP incorporation
results
shocked
indicate cells
reflects
DNA synthesis.
When a temperature ref.
x g,
with
activity.
8) was shifted
and TTP incorporation
sensitive to
45 C for was then
DNA initiation 45 minutes measured
792
mutant
prior at
to cold
45 C, the
(61304, shock,
same amount
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
50-
Figure 2. g. subtilis
of 3)
[%]TTP strain
incorporated into TCA precipitable 60015 (see Materials and Methods).
incorporation Figure
l
Moreover,
was observed 3 again
as in
shows the
TTP incorporation
inhibitor
in
61304
These
results
cells
reflected
To verify cold
shocked
cells
60439,
(MfG),
incorporated
contrast,
of
sensitive
strain
in g.
subtilis
TTP incorporation
several
repair
to W but
to methyl
TTP at least
as well
60437,
is
which
793
is a (9).
cold
shocked
synthesis.
was examined
deficient not
which
in
replication
by ATP.
to reduced
31,
than
as the
sensitive
by
(Figure
reaction
Figure
TTP incorporation rather
conclusion,
Strain
In
that
DNA repair
this
of the
DNA replication
suggested
strain
was insensitive
(HPUra,
of
standard
stimulation
6-(p-hydroxyphenylazo)-uracil specific
the
material
mutants
in (Figure
methane
sulfonate
standard
strain.
to both
W
4).
and MMS,
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
RESEARCH, COMMUNICATIONS
TABLE 3
Contents
of
Complete II
Reaction
107
II
+ thymidine*
110
If
+ TMP*
83
1,
+ TDP*
80
II
+ TTP*
6
11
- MgS04
11
11
- ATP
15
II
- ATP + adenosine/AMP/ADP**
17-33
11
- ATP + CTP**
55
**2
UM
the
lower
10% of
the
Strauss,
(data
not
levels
as well
as the
absorbing
All
conclusion
incorporation
61477,
the
which
has less
on the
strain
than
incorporated
based
has
TTP very
were permeabilized
strain,
which
parental
incorporated
(9),
strains
standard
even
poorly 0.5% of
less
by cold comparison
TTP
shock of W
released. that
reflects
in
the mixture.
cold
shocked
DNA repair
may explain
37 C before
of
61476,
I activity
these
We conclude incorporation
I activity
The mutant
material
The strain
communication)
Polymerase
4).
of TTP.
Polymerase
shown).
normal
mM
personal
(Figure
at
Incorporated
+ thymine"
incorporated
(B.
% i3H]TTP
100
*165
-nly
Mixture
addition
the
rather
necessity of
cells
cold
Incubation
of B. - subtilis than replication.
for shocked
the
15 minute
cells
at 0 C or in
794
to the the
TTP This incubation TTP
presence
of
Vol. 55, No. 3,1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIQNS
MINUTES
Figure 3. [%]TTP incorporated cold shocked (1) C) cells of g. and Methods).
p-hydroxymercuribenzoate incorporation
(Figure
to DNA is
a prerequisite
tion
of
repair
been
reported
ATP-dependent
5).
37 C reduced Presumably,
for
by activation in E.
(3).
trations,
which
This
of
could
explain
subsequent
repair
endogenous with
in g. is
material by (see Materials
TTP
nuclease-mediated
permeabilized
enzyme
the
subsequent
deoxyribonuclease
reported
by
at
into TCA precipitable subtilis strain 61304
synthesis.
nucleases
inhibited
the
stimulation
Induc-
has previously
Tris-EDTA
subtilis
also
damage
(1,2).
has recently by high
An been
ATP concen-
of TTP incorporation
ATP. In
repair allowed
contrast deficient incorporation
to cold
shocking,
mutants
(60437 of TTP,
permeabilization and 61476)
which
795
with
was sensitive
of toluene
the (7)
to HPUra
two
BIOCHEMICAL
Vol. 55, No. 3, 1973
AND BIOPHYSICAL
5
20-
9 a’ 6 E 8 z
Prevncubated
/
;
IO-
/ /
I 15 MINUTES
I 20
MINUTES
Figure 4. L3HlTTP incorporated cold shocked (0 C) cells of the (see Materials and Methods).
and therefore
represented
had been
shocked
about
50% less
treatment. by the This
Possibly, cold
could
fraction replicate
TTP than
shock
(50%) of their
only
that the
the
cells
and the
imply
into TCA precipitable subtilis strain 60015
DNA replication. before
toluene
which about
other
in
treatment
received
cells
DNA. 796
which
the
toluene
were affected
50% permeabilized
such a way that
cells
incorporated
only
by cold
by
material by (see Materials
However,
50% of the
permeabilization cells
30
into TCA precipitable material indicated strain of II. subtilis
Figure 5. [3H]TTP incorporated cold shocked (0 C) cells of g. and Methods).
cold
at 37OC
/
2
I IO
RESEARCH COMMUNICATIONS
by toluene. shock they
affects
a
can no longer
BIOCHEMICAL
Vol. 55, wo. 3,1973
sure
It
is
to
low temperatures,
refrigerated this
should
apparent
that
centrifuge, be considered
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
permeabilization
of B_. subtilis
as may inadvertently can affect in the
several design
occur cellular
by
expo-
in a processes;
of experiments.
REFERENCES 1.
Buttin,
G.,
and Kornberg,
A.
(1966)
J.
Biol.
Chem., 241,
5419-
5427. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11.
Buttin, G., and Wright, M. (1968) Cold Spring Harbor Symp. 33, 259-269. Chestukhin, A. V., Shemykin, M. F., Kalinina, N. A., and Prozorov, A. A. (1972) FEBS Letters 24, 121-125. Esfahani, M., Limbrick, A. R., Knutton, S., Oka, T., and Wakil, J. (1971) Proc. Nat. Acad. Sci. USA 68, 3180-3184. Farrell, J., and Rose, A. H. (1967) in Therzbiology, A. H. Academic Press, New York. Rose, ed., pp. 147-218, Fortnagel, P., and Freese, E. (1968) J. Bacterial. 95, 14311438. Matsushita, T., White, K. P., and Sueoka, N. (1971) Nature New Biol. 232, 111-114. Mendelson, N. H., and Gross, J. D. (1967) J. Bacterial. 2, 1603-1608. Neville, M., and Brown, N. C. (1972) Nature New Biol. 240, 80-82. Rhinehart, K. V., and Copeland, J. C. (1973) Biochim. mphys. Acta 294, l-7. W. H. (1967) Biochem. Biophys. Res. Smeatc J. R., and Elliott, Commun. 26, 75-81.
797