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ATP-independent, DNA synthesis by DNA polymerase II in toluenized Bacillus subtilis
BIOCHEMICAL
Vol. 60, No. 4,1974
ATP-INDEPENDENT,
DNA
IN
AND BIOPHYSICAL
SYNTHESIS
TOLUENIZED Harvard
Department
of
Received
August
SUMMARY: merase II increases normal DNA merase III.
DNA an
ATP
as or
ADP
serve
found
in
ulate
that
that
it III
merases
from
--in
their the
We have
previously
of
and
exonucleases,
cells
(7).
taining
DNA
Since
polymerase
at
the
Medical
Center
60680
and
to
that
an
might
agent
effects
the also
the
same
polymerase
specway
III,
that
b.reaks
with DNA which
could in
of
polymer-
act
in
One
activities form
DNA
have
without
breaks (5).
case,
or
ATP
produce
to
function
that
complexed
either
on1 y two
III,
postulated
by polyalso that of by poly-
frees in
the
poly-
the DNA,
polymight
ATP-requirement. that
the
antibiotic
complexes,
that
the
previous
experiments
II
it
I appears
the
polymerases
the
forms,
and
we
Yet
polymerase
In
cell-membrane
paper
the
synthesis of ATP between synthesis
polymerase
2).
cells
of
shown
as
lack
(1,
been
initiate
polymerase
I,
has
(6).
was
describe
Illinois
that
ADP
for
to
vitro
polymerases
Copyright All rights
toluenized
activity
three
this
in
complexed
eliminate
sociation
of
polymerases
It
act the
or
4).
sites
might
star,
ATP cells,
initiation
initiates
merase
also
(3,
nucleases
ATP
cells
for these
vitro
as
University Chicago, Illinois
toluenized
rement
ATP-dependent then
in
requi
in
II
Rei ter
stimulates ATP-independent DNA repair B. subtilis cells. In the presence with BrdUTP, of DNA with a density DNA, and it enhances replicative DNA
synthesis
yet
POLYMERASE
SUBTILIS
22,1974
Phleomycin in toluenized the synthesis, and hybrid
absolute
ases
Microbiology,
BY DNA
BACILLUS
RESEARCH COMMUNICATIONS
it
well
that
greatly
responsible
II
and
Ill, the
pol
point.
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
with
was
increased A-
1371
strains
both
synthesis
done it
causes
activates
DNA were
for using
it
enhances
experiments as
phleomycin
of
in
the
endonucleases toluenized
wild-type
not
clear
DNA
synthesis.
1.
subtilis
dis-
cells
which
to
con-
of
the
In
this clarify
Vol. 60, No. 4, 1974
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS The cribed
strains and
sitivity tion
to their
8.
subtilis
given
to
us
UV
irradiation.
viability
bility
of
168 thy-
cribed
in
detail
F2
by
Dr.
fell uvr+ in
the
and
Cozzarelli
After
a 30
1.5
to
cells. of
in
untreated,
and
in
as
these
second, in
400
had
to
used
the
erg/mm’
contrast in
each
were expected
dose
an
des-
of
80% loss
senirradiaof
experiment
via-
are
des-
figure.
DISCUSSION
controls,
the F2
experiments
They
appropriate
AND
I
in
(8).
methods the
UV-irradiated
METHODS
used
2 logs, The
legend
established,
F22
N.
RESULTS We first
AND
RESEARCH COMMUNICATIONS
characteristics
cells.
Figure
of 1 shows
DNA that
synthesis a
very
small
I
IO
20
30 40 MINUTES
50
60
Figure 1. DNA synthesis in toluenized control and UV-irradiated cells. 8. medium (Spizizzin’s minimal medium plus 0.5% subti 1 is Fq was grown in CHT20 glucose, 0.04% acid-hydrolysed casein, and 20 @g/ml tryptophan) to a concentration of 1 x lo8 cells/ml. Fifty ml of cells were washed and resuspended in 10 One of ml of cold O.D5 M KH2P04 pH 7.4, and divided into two 5 ml ali uots. 9) . these was i rradiated with UV 1 ight for 60 seconds (800 ergs/mm Both samples were then centrifuged, resuspended in 0.6 ml of cold basic buffer (0.013 M MgS04, 0.002 M dithiothreitol, 0.07 M KH2PO4, pH 7.4) and warmed to 37 C for 30 seconds. They were then treated with 0.04 ml toluene for 2.5 min and chilled One-tenth ml of toluenized cells was added to 0.4 ml of cold reaction to 4 c. mix, and DNA synthesis was started by transfer to 37 C. The reaction mix was 30 PM dCTP, 10 PM dGTP, 30 WM dATP.5 ELM-16 DC 3H-dTTP, in basic buffer. When present, N-ethylrnaleimide (NEM) was 10.6 mM and ATP was 1.5 mM. DNA synthesis in 50 ~1 samples that was measured by determining the amounts of 1 H activity precipitated in 5% cold trichloroacetic acid (TCA).
1372
Vol. 60, No. 4,1974
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
amount
occurred
both
the
of
also
DNA
shows
(NEM)
so
synthesis
that that
this it
polymerase.
synthesis
3 to
F2
ccl
and
UV
irradiation
1 s had
phleomycin
these
no
to
by
the ATP
cells,
to
5 to
in
ATP
when
ATP
as
NEM-
cells.
expected, I)
was
by
irradiated
Thus,
It
increased
(polymerase
only
ATP.
maleimide
mixes
6-fold
increases.
synthesis
of
residual
reaction
NEM-resistant
DNA
absence
mM N-ethyl of
the
and
ATP-stimulated
ATP-independent,
10.6
utilization
of
untreated
in
eliminated
addition
enhanced
measured just
amounts
cells
in
due
cultures
added
activity, to
the
mix. We next
the
The
blocked
the
was
probably
b-fold
NEM completely
reaction
synthesis
was
sensitive
in
of
same made
ONA
before
synthesis as
as
much
cells
more
cells unlike
ONA
IO
treated
Figure
untreated
However, or
in
toluenization. in
before.
synthesis
as
20
in UV-i
was
30 40 MINUTES
2 (bottom) this
in
50
3 min
with
16 pg/ml
shows
that
the
experiment
rradiated
made
for
cells, untreated
were
essentially
phleomycin-treated cells
plus
ATP.
In
60
Fiqure 2. DNA synthesis in toluenized control and phleomycin-treated cells. Fifty ml of cells were washed with cold 0.05 M KH2PO pH 7.4, resuspended in 1.2 ml of cold basic buffer, and divided’ into two 0. 2 ml samples. Phleomycin (16 ug/ml) was added to one sample. Both samples were incubated at 37 C for 3 min, then shaken gently with 0.04 ml toluene for 2.5 minutes, and chilled to 4 c. One-tenth ml samples were added to reaction mixes and DNA synthesis measured as before (legend of Figure 1). Where present, 6-(p-hydroxyphenylaze)uracile (HPUra) was 100 PM.
1373
Vol. 60, No. 4, 1974
this
experiment
DNA made times
as
the
in
well
DNA
a
ATP,
than
inhibition
amounts
was
and
NEM
those
completely
In
other
complete.
between
those
in
polymerase
II
activity
NEM,
at
IO mM NEM
inhibition
polymerase
II
determine
less
unt/l
mycin
stimulates In
order
synthesis ence
to
was of
due
III
showing
100
toluenized in
these
thesis
in
untreated
is
the
pol
I I .
in Thus,
order
synthesis
we
containing cells,
the
if
measured
the
absence
and
the
NEM
data
is
due
ATP,
in
of place
III
activity.
the
of
incorporated
a very
1374
vitro,
5 f&l and of
that
the
phleo-
HPUra
HPUra
data
the
small
Is
Synsyn-
same 9
by
in
to
amount 18%.
This
eliminated was
due
suggest
to
that
the
activity. repair
synthesized Figure
ccl
(8).
were
synthesis
pres-
reduced
only
DNA
II
F2
III
by
the
synthesis
activity
stimulated
dTTP.
ATP
inhibits
However,
polymerase
DNAs
to
in
in
that
III
and
with
concentrations suggest
cells
the
activity,
DNA
2 shows
increased
used
plus
specifically
40%.
to
II
polymerase
phleomycin
densities
BdUTP, of
of
in
happened
polymerase
in
synthesis
Since,
data
affect
approximately
15
occurred
synthesis
which this
Figure
to
ceils,
cells
measured
HPUra-resistant
normal
whether the
we
of
phleomycin-stimulated
phleomycin-treated
all
both
determine
3H-dATP in
in
synthesis to
our
significantly
5%.
by
which
phleomycin-stimulated In
cells
expected cells
ymerase
0 to
synthesis
reduction
not
treated
DNA
higher
whether
that
having
fell F2
reduced
treated
cells
cells
at
(HPUra)
did
reduced
polymerase
III,
10
8 mM NEM was
polymerase
(g),
ambiguously or
the that
alone.
not
total
We confirmed
F22
ATP
amount
made
phleomycin-treated
than
II
in
which
inhibited
rather
the
they
synthesis
8 mM NEM
but
is
polymerase
UM HPUra
strain
thesis
from
to
the
plus III
5 times
ATP.
in
in
increasingly
activity.
that
HPUra
made
6-(p-hydrooxyphenylase)-uracil
polymerase
of
is
minus
of
The
polymerase
almost
synthesis
all,
cells
RESEARCH COMMUNICATIONS
experiments
experiments
control
inhibits
other cells
not
as
made
made
ATP-stimulated but
not
In
control
ail
ATP.
intermediate
alone,
in
fraction,
minus
ATP
ATP.
made
blocked
AND BIOPHYSICAL
minus
minus
was
large
cells
cells
cells
(10.6’mM) as
treated
treated
control
more NEM
BIOCHEMICAL
or in
reaction
3 shows amount
replicative
that of
mixes control
3H into
DNA
Vol.
60,
No.
of
normal
density
of
repair
synthesis.
3H
into
into
DNA DNA
activity taining
(fractions
of hybrid
in
fraction
imately The
they
presence 4
of
times
plus-ATP in
of
cells
hybrid
ATP,
expected
DNA).
In
location in
6
performed
there
Clearly, well
the
absence
of
of
3H
repair
of
the
23),
of
con-
ATP,
the
into
control in
of
synthesis
occurred
and with
the
nor-
approxATP.
phleomycin-
replicative
synthesis
in
occurred
of
synthesis.
cells
14)
phleo-
DNA
repair
and
radio-
strands
synthesis
reinitiation
synthesis
to
sign
two
repair
13 and
repair
as
DNA with
than
was
only
of
more
(fractions
no
amount
incorporated 21
is
amount
times
a small cells
There
amount
synthesis DNA
performed
(fractions
18).
to
COMMUNKATIONS
control
density
a significant
that
as
these
a significant
heavy-heavy
replicative
ATP,
contrast,
replicative
strands.
and
the
they
indicates
is,
normal
RESEARCH
they
That
16
performed
ATP
more
appearance
than
incorporated
a,
of
(fractions
13,
BLOPHYSICAL
26).
presence
density
(heavy-heavy
density,
AND
to
greater
cells
the
the
slightly
mycin-treated
In
24
In
of
BU
mal
BIOCHEMICAL
4,1974
in
the
absence
of
the
presence
of
was
mediated
by
ATP. In
order
polymerase HPUra
II or
in
merase less
or
III,
intact.
we
by the
synthesis
in
polymerase
I I I.
stimulated
directly
II
which
in
is
of
in
the
is
heavier
some
turn
as
the
1.756
(insert)
the
Figure
this
by
phleomycin the
activity
had
1.703
gm/cm3
density
gm/cm3
density
of
hybrid
1375
of
polymore
total
DNA
compared
case,
to
to
polymerase
have III
replimediated
activity
was
polymerase
In
this
DNA made
by
polymerase
a density
of
1.719
of
and
of
the
normal DNA.
This
the
been
stimulated III.
about repair
intermediate
or
syn-
ATP-dependent
appears
polymerase
part 23)
of
phleomycin
of
,uM
synthesis
as
any
this
whether
a significant (fraction
In
100
inhibitors
inhibition
2.
of
repair
experiment
whether or
presence
both
left
cells
clear
the
that
greater
phleomycin-treated not
in
but
the
of
is
ATP
made
synthesis for
synthesis
shows
in
It
that of
than
the
DNAs
measured
experiment
increased
interest
presence
3
account HPUra
replicative
the
replicative
NEM and in
the
examined
Figure
We cannot
by
cative
whether
5 mM NEM.
measurements
heavy
establish
II I eliminated
thesis
by
to
DNA,
regard, II
gm/cm3. but
it
This not
density
as
Vol.
60,
No.
4, 1974
BIOCHEMICAL
AND
BIOPHYSICAL
i
‘L,.
RESEARCH
l
COMMUNICATIONS
PHI.EO+ATP
A CONTROL+ ATP
If
FRkTlON
i!
i
, 0’
1
I
5
IO
15 20 FRACTION
25
30
35
Fiqure 3. CsCl density gradients of DNA synthesized in toluenized control and phleomycin-treated ccl Is, in the presence of bromodeoxyuridine triphosphate (BrdUTP) . One-tenth ml samples of cells, treated with phleomycin and toluene, as described in the legend of Figure 2, were added to 0.4 ml of reaction mix, which contained 40 JJM BrdUTP in place of dTTP, and 6 uM-I7 tic 3 H dATP in place of 30 uM dATP. After 28 min at 37 C, 0.2 ml of cells was added to 0.2 ml of 0.05 M EDTA-0.05 Tris pH 8.1, plus 200 ug/ml lysozyme. After 5 min at 37 C, 0.02 ml of 10 mg/ml pronase was added for 2 min, and then 0.02 ml of 1% SOS was added. The lysates were sonicated with the microtip attachment to the Two-tenths ml of Bronson Sonifier, at a power setting of 5, for I.5 min. lysate was added to CsCl in EDTA-Tris, plus 10 mM NaCl. A 0.02 ml sample containing 1000 cpm, l&C-labeled, normal density 8. subtilis DNA was added to each tube, and the CsCl concentration was adjusted to a refractive index of 1.4022. The samples were centrifuged for 48 hrs at 37,000 rpm, at 18 C in a Spinco SW50.1 head. Forty-two, eight-drop fractions were collected from the bottom of the tubes, and the amounts of TCA-precipitable 3H activity in each fraction were determined. A separate tube containing only CsCl, adjusted to a refractive index of 1.4022 was run with each set. The refractive indices of each eightdrop fraction from these tubes were measured and found to change an average of O.O007/fraction. This is equivalent to a density than e of approximately 0.008 gm/cm3/fraction. The arrow shows the position of )&-labeled, normaldensity DNA. The inserted qraph shows the distribution in CsCl gradients, or DNA made in phleomycin-treated cells in the presence of 5 M NEM, or 100 M HPUra.
DNA may cells
be (IO),
the or
equivalent of
the
of single
methane-sulfonate-treated The
experiments
the strand
ccl leave
“long
Is
open
patch”
repair
containing
DNA
made that
in
polymerase
accumulates
in
(1 I). the
question
1376
of
how
phleomycin
l-
stimulates
methyl
BIOCHEMICAL
Vol. 60, No. 4, 1974
polymerase endonuclease as
I l-induced
initiation
gests
the
dAT-rich
(7,
also
merase
is
(13,
14)
to
DNA
I I activity
of
arrest
the
of
cells
DNA
it
does
but
that
the
supplies
a to
II dAT-rich
DNA
templates
apparent
cells
these
activities
are and
(Reiter,
of
number
of
way
to
potentiate seen
triphosphates
toluenized
interacts
may
in
proteins poly-
single-stranded rapid,
in
whole
cells
that
are
available
complete
synthesis
in
acts II
on
and
III
because the
toluenized
whole
cells
activities,
they
to
(16).
regions
the
polymerase
at
molecule
enhance
phleomycin
be DNA
the
DNA-unwinding
stimulating
sug-
with
breaks
causing
that
cells It
specifically long
while
possible
not
as
phleomycin
DNA serve
specific. that
and
is
the
(12)
same
nor
unpublished),
in
containing
cells
It
toluenized
the
breaks,
relatively
regions,
whole
on
be
limited
in
anomaly in
ATP
DNA synthesis
a
RESEARCH COMMUNICATIONS
UV-induced
polypeptide
cause
unexplained.
of
template
small
at
synthesis
remains
neither
must
polymerase
bind
Finally,
in
stimulus
which
stimulates
IS),
that
ATP-independent
phleomycin
regions
which
as
for
phleomycin,
fact
breaks
sites
that
that
The
I I activity.
AND BIOPHYSICAL
do
not
have
toluenized
cells. ACKNOWLEDGMENT: Research Grant
This No.
investigation CA-17123-03Al
was from
the
supported National
by Public Cancer
Health Institute.
Service
REFERENCES
I. 2. Z:
Pisetsky, Biol. & Virasoro, Kornberg, Moses, R.
2, 5.
D.,
Berkower,
I .,
Wickner,
R.,
and
Hurwitz,
J.
(1972)
J.
Med.
557-571. and Hirota, Y. (1974) Biochimie 56, 363-371. M. L. (1971) Proc. Nat. Acad. Sci. 68, 761-764. C. C. (1971) Biochem. Biophys. Res. Commun.
Mardoh, J. and Gefter, and Richardson,
S., T. E.
1565-1971.
6. 7.
Masler, 243. Wickner, Reiter,
8.
Cozzarell
W.,
Hamewalt,
P.
W. H.,
and Komberg, Milewskiy,
M.
and
Shizuya,
A. (1973) and Kelley,
H.
(1973)
P.
Nat. (1972)
Proc.
Nature Acad. J.
New
Biol.
Sci. s, Bacterial.
2&
242-
3679-3683. fi, 586-
592. 2, 9. 10. 11. 12.
R.
and
Low,
R.
L.
(1973)
Biochem.
Biophys.
Res.
Commun.
Gass, K. B. and Cozzarelli, N. R. (1973) In Methods in Enzymology, Grossman, L. and Moldave, K., Academic Press, New York. Cooper, P. K. and Hanawalt, P. C. (1972) J. Mol. Biol. 67, l-10. Kato, K. and Strauss, 8. (1974) Proc. Nat. Acad. Sci. 71, 1969-1973. Takita, T., Mursoka, Y. and Yoshioka, T. (1972) J. Antziol. (Tokyo)
A. 5, 13. 14. 15. 16.
i , N.
151-157.
Falaschi, Pietsch, Farrell, Sigai, Proc.
eds.
Ser.
755-758. A. and Kornberg, A. (1964) Fed. P. and Garrett, H. (1968) Nature L. and Reiter, H. (1973) Antimicrob. N., Delius, H., Kornberg, T., Gefter, Nat. Acad. Sci. 69, 3537-3541.
1377
Proc. 23, 440-445. (Lond.rm, 488-489. Ag. Chemother. fr, M. L. and Alberts,
320-326. B. (1972)
Vol. 60, No. 4,1974
ATP-INDEPENDENT,
DNA
IN
AND BIOPHYSICAL
SYNTHESIS
TOLUENIZED Harvard
Department
of
Received
August
SUMMARY: merase II increases normal DNA merase III.
DNA an
ATP
as or
ADP
serve
found
in
ulate
that
that
it III
merases
from
--in
their the
We have
previously
of
and
exonucleases,
cells
(7).
taining
DNA
Since
polymerase
at
the
Medical
Center
60680
and
to
that
an
might
agent
effects
the also
the
same
polymerase
specway
III,
that
b.reaks
with DNA which
could in
of
polymer-
act
in
One
activities form
DNA
have
without
breaks (5).
case,
or
ATP
produce
to
function
that
complexed
either
on1 y two
III,
postulated
by polyalso that of by poly-
frees in
the
poly-
the DNA,
polymight
ATP-requirement. that
the
antibiotic
complexes,
that
the
previous
experiments
II
it
I appears
the
polymerases
the
forms,
and
we
Yet
polymerase
In
cell-membrane
paper
the
synthesis of ATP between synthesis
polymerase
2).
cells
of
shown
as
lack
(1,
been
initiate
polymerase
I,
has
(6).
was
describe
Illinois
that
ADP
for
to
vitro
polymerases
Copyright All rights
toluenized
activity
three
this
in
complexed
eliminate
sociation
of
polymerases
It
act the
or
4).
sites
might
star,
ATP cells,
initiation
initiates
merase
also
(3,
nucleases
ATP
cells
for these
vitro
as
University Chicago, Illinois
toluenized
rement
ATP-dependent then
in
requi
in
II
Rei ter
stimulates ATP-independent DNA repair B. subtilis cells. In the presence with BrdUTP, of DNA with a density DNA, and it enhances replicative DNA
synthesis
yet
POLYMERASE
SUBTILIS
22,1974
Phleomycin in toluenized the synthesis, and hybrid
absolute
ases
Microbiology,
BY DNA
BACILLUS
RESEARCH COMMUNICATIONS
it
well
that
greatly
responsible
II
and
Ill, the
pol
point.
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
with
was
increased A-
1371
strains
both
synthesis
done it
causes
activates
DNA were
for using
it
enhances
experiments as
phleomycin
of
in
the
endonucleases toluenized
wild-type
not
clear
DNA
synthesis.
1.
subtilis
dis-
cells
which
to
con-
of
the
In
this clarify
Vol. 60, No. 4, 1974
BIOCHEMICAL
AND BIOPHYSICAL
MATERIALS The cribed
strains and
sitivity tion
to their
8.
subtilis
given
to
us
UV
irradiation.
viability
bility
of
168 thy-
cribed
in
detail
F2
by
Dr.
fell uvr+ in
the
and
Cozzarelli
After
a 30
1.5
to
cells. of
in
untreated,
and
in
as
these
second, in
400
had
to
used
the
erg/mm’
contrast in
each
were expected
dose
an
des-
of
80% loss
senirradiaof
experiment
via-
are
des-
figure.
DISCUSSION
controls,
the F2
experiments
They
appropriate
AND
I
in
(8).
methods the
UV-irradiated
METHODS
used
2 logs, The
legend
established,
F22
N.
RESULTS We first
AND
RESEARCH COMMUNICATIONS
characteristics
cells.
Figure
of 1 shows
DNA that
synthesis a
very
small
I
IO
20
30 40 MINUTES
50
60
Figure 1. DNA synthesis in toluenized control and UV-irradiated cells. 8. medium (Spizizzin’s minimal medium plus 0.5% subti 1 is Fq was grown in CHT20 glucose, 0.04% acid-hydrolysed casein, and 20 @g/ml tryptophan) to a concentration of 1 x lo8 cells/ml. Fifty ml of cells were washed and resuspended in 10 One of ml of cold O.D5 M KH2P04 pH 7.4, and divided into two 5 ml ali uots. 9) . these was i rradiated with UV 1 ight for 60 seconds (800 ergs/mm Both samples were then centrifuged, resuspended in 0.6 ml of cold basic buffer (0.013 M MgS04, 0.002 M dithiothreitol, 0.07 M KH2PO4, pH 7.4) and warmed to 37 C for 30 seconds. They were then treated with 0.04 ml toluene for 2.5 min and chilled One-tenth ml of toluenized cells was added to 0.4 ml of cold reaction to 4 c. mix, and DNA synthesis was started by transfer to 37 C. The reaction mix was 30 PM dCTP, 10 PM dGTP, 30 WM dATP.5 ELM-16 DC 3H-dTTP, in basic buffer. When present, N-ethylrnaleimide (NEM) was 10.6 mM and ATP was 1.5 mM. DNA synthesis in 50 ~1 samples that was measured by determining the amounts of 1 H activity precipitated in 5% cold trichloroacetic acid (TCA).
1372
Vol. 60, No. 4,1974
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
amount
occurred
both
the
of
also
DNA
shows
(NEM)
so
synthesis
that that
this it
polymerase.
synthesis
3 to
F2
ccl
and
UV
irradiation
1 s had
phleomycin
these
no
to
by
the ATP
cells,
to
5 to
in
ATP
when
ATP
as
NEM-
cells.
expected, I)
was
by
irradiated
Thus,
It
increased
(polymerase
only
ATP.
maleimide
mixes
6-fold
increases.
synthesis
of
residual
reaction
NEM-resistant
DNA
absence
mM N-ethyl of
the
and
ATP-stimulated
ATP-independent,
10.6
utilization
of
untreated
in
eliminated
addition
enhanced
measured just
amounts
cells
in
due
cultures
added
activity, to
the
mix. We next
the
The
blocked
the
was
probably
b-fold
NEM completely
reaction
synthesis
was
sensitive
in
of
same made
ONA
before
synthesis as
as
much
cells
more
cells unlike
ONA
IO
treated
Figure
untreated
However, or
in
toluenization. in
before.
synthesis
as
20
in UV-i
was
30 40 MINUTES
2 (bottom) this
in
50
3 min
with
16 pg/ml
shows
that
the
experiment
rradiated
made
for
cells, untreated
were
essentially
phleomycin-treated cells
plus
ATP.
In
60
Fiqure 2. DNA synthesis in toluenized control and phleomycin-treated cells. Fifty ml of cells were washed with cold 0.05 M KH2PO pH 7.4, resuspended in 1.2 ml of cold basic buffer, and divided’ into two 0. 2 ml samples. Phleomycin (16 ug/ml) was added to one sample. Both samples were incubated at 37 C for 3 min, then shaken gently with 0.04 ml toluene for 2.5 minutes, and chilled to 4 c. One-tenth ml samples were added to reaction mixes and DNA synthesis measured as before (legend of Figure 1). Where present, 6-(p-hydroxyphenylaze)uracile (HPUra) was 100 PM.
1373
Vol. 60, No. 4, 1974
this
experiment
DNA made times
as
the
in
well
DNA
a
ATP,
than
inhibition
amounts
was
and
NEM
those
completely
In
other
complete.
between
those
in
polymerase
II
activity
NEM,
at
IO mM NEM
inhibition
polymerase
II
determine
less
unt/l
mycin
stimulates In
order
synthesis ence
to
was of
due
III
showing
100
toluenized in
these
thesis
in
untreated
is
the
pol
I I .
in Thus,
order
synthesis
we
containing cells,
the
if
measured
the
absence
and
the
NEM
data
is
due
ATP,
in
of place
III
activity.
the
of
incorporated
a very
1374
vitro,
5 f&l and of
that
the
phleo-
HPUra
HPUra
data
the
small
Is
Synsyn-
same 9
by
in
to
amount 18%.
This
eliminated was
due
suggest
to
that
the
activity. repair
synthesized Figure
ccl
(8).
were
synthesis
pres-
reduced
only
DNA
II
F2
III
by
the
synthesis
activity
stimulated
dTTP.
ATP
inhibits
However,
polymerase
DNAs
to
in
in
that
III
and
with
concentrations suggest
cells
the
activity,
DNA
2 shows
increased
used
plus
specifically
40%.
to
II
polymerase
phleomycin
densities
BdUTP, of
of
in
happened
polymerase
in
synthesis
Since,
data
affect
approximately
15
occurred
synthesis
which this
Figure
to
ceils,
cells
measured
HPUra-resistant
normal
whether the
we
of
phleomycin-stimulated
phleomycin-treated
all
both
determine
3H-dATP in
in
synthesis to
our
significantly
5%.
by
which
phleomycin-stimulated In
cells
expected cells
ymerase
0 to
synthesis
reduction
not
treated
DNA
higher
whether
that
having
fell F2
reduced
treated
cells
cells
at
(HPUra)
did
reduced
polymerase
III,
10
8 mM NEM was
polymerase
(g),
ambiguously or
the that
alone.
not
total
We confirmed
F22
ATP
amount
made
phleomycin-treated
than
II
in
which
inhibited
rather
the
they
synthesis
8 mM NEM
but
is
polymerase
UM HPUra
strain
thesis
from
to
the
plus III
5 times
ATP.
in
in
increasingly
activity.
that
HPUra
made
6-(p-hydrooxyphenylase)-uracil
polymerase
of
is
minus
of
The
polymerase
almost
synthesis
all,
cells
RESEARCH COMMUNICATIONS
experiments
experiments
control
inhibits
other cells
not
as
made
made
ATP-stimulated but
not
In
control
ail
ATP.
intermediate
alone,
in
fraction,
minus
ATP
ATP.
made
blocked
AND BIOPHYSICAL
minus
minus
was
large
cells
cells
cells
(10.6’mM) as
treated
treated
control
more NEM
BIOCHEMICAL
or in
reaction
3 shows amount
replicative
that of
mixes control
3H into
DNA
Vol.
60,
No.
of
normal
density
of
repair
synthesis.
3H
into
into
DNA DNA
activity taining
(fractions
of hybrid
in
fraction
imately The
they
presence 4
of
times
plus-ATP in
of
cells
hybrid
ATP,
expected
DNA).
In
location in
6
performed
there
Clearly, well
the
absence
of
of
3H
repair
of
the
23),
of
con-
ATP,
the
into
control in
of
synthesis
occurred
and with
the
nor-
approxATP.
phleomycin-
replicative
synthesis
in
occurred
of
synthesis.
cells
14)
phleo-
DNA
repair
and
radio-
strands
synthesis
reinitiation
synthesis
to
sign
two
repair
13 and
repair
as
DNA with
than
was
only
of
more
(fractions
no
amount
incorporated 21
is
amount
times
a small cells
There
amount
synthesis DNA
performed
(fractions
18).
to
COMMUNKATIONS
control
density
a significant
that
as
these
a significant
heavy-heavy
replicative
ATP,
contrast,
replicative
strands.
and
the
they
indicates
is,
normal
RESEARCH
they
That
16
performed
ATP
more
appearance
than
incorporated
a,
of
(fractions
13,
BLOPHYSICAL
26).
presence
density
(heavy-heavy
density,
AND
to
greater
cells
the
the
slightly
mycin-treated
In
24
In
of
BU
mal
BIOCHEMICAL
4,1974
in
the
absence
of
the
presence
of
was
mediated
by
ATP. In
order
polymerase HPUra
II or
in
merase less
or
III,
intact.
we
by the
synthesis
in
polymerase
I I I.
stimulated
directly
II
which
in
is
of
in
the
is
heavier
some
turn
as
the
1.756
(insert)
the
Figure
this
by
phleomycin the
activity
had
1.703
gm/cm3
density
gm/cm3
density
of
hybrid
1375
of
polymore
total
DNA
compared
case,
to
to
polymerase
have III
replimediated
activity
was
polymerase
In
this
DNA made
by
polymerase
a density
of
1.719
of
and
of
the
normal DNA.
This
the
been
stimulated III.
about repair
intermediate
or
syn-
ATP-dependent
appears
polymerase
part 23)
of
phleomycin
of
,uM
synthesis
as
any
this
whether
a significant (fraction
In
100
inhibitors
inhibition
2.
of
repair
experiment
whether or
presence
both
left
cells
clear
the
that
greater
phleomycin-treated not
in
but
the
of
is
ATP
made
synthesis for
synthesis
shows
in
It
that of
than
the
DNAs
measured
experiment
increased
interest
presence
3
account HPUra
replicative
the
replicative
NEM and in
the
examined
Figure
We cannot
by
cative
whether
5 mM NEM.
measurements
heavy
establish
II I eliminated
thesis
by
to
DNA,
regard, II
gm/cm3. but
it
This not
density
as
Vol.
60,
No.
4, 1974
BIOCHEMICAL
AND
BIOPHYSICAL
i
‘L,.
RESEARCH
l
COMMUNICATIONS
PHI.EO+ATP
A CONTROL+ ATP
If
FRkTlON
i!
i
, 0’
1
I
5
IO
15 20 FRACTION
25
30
35
Fiqure 3. CsCl density gradients of DNA synthesized in toluenized control and phleomycin-treated ccl Is, in the presence of bromodeoxyuridine triphosphate (BrdUTP) . One-tenth ml samples of cells, treated with phleomycin and toluene, as described in the legend of Figure 2, were added to 0.4 ml of reaction mix, which contained 40 JJM BrdUTP in place of dTTP, and 6 uM-I7 tic 3 H dATP in place of 30 uM dATP. After 28 min at 37 C, 0.2 ml of cells was added to 0.2 ml of 0.05 M EDTA-0.05 Tris pH 8.1, plus 200 ug/ml lysozyme. After 5 min at 37 C, 0.02 ml of 10 mg/ml pronase was added for 2 min, and then 0.02 ml of 1% SOS was added. The lysates were sonicated with the microtip attachment to the Two-tenths ml of Bronson Sonifier, at a power setting of 5, for I.5 min. lysate was added to CsCl in EDTA-Tris, plus 10 mM NaCl. A 0.02 ml sample containing 1000 cpm, l&C-labeled, normal density 8. subtilis DNA was added to each tube, and the CsCl concentration was adjusted to a refractive index of 1.4022. The samples were centrifuged for 48 hrs at 37,000 rpm, at 18 C in a Spinco SW50.1 head. Forty-two, eight-drop fractions were collected from the bottom of the tubes, and the amounts of TCA-precipitable 3H activity in each fraction were determined. A separate tube containing only CsCl, adjusted to a refractive index of 1.4022 was run with each set. The refractive indices of each eightdrop fraction from these tubes were measured and found to change an average of O.O007/fraction. This is equivalent to a density than e of approximately 0.008 gm/cm3/fraction. The arrow shows the position of )&-labeled, normaldensity DNA. The inserted qraph shows the distribution in CsCl gradients, or DNA made in phleomycin-treated cells in the presence of 5 M NEM, or 100 M HPUra.
DNA may cells
be (IO),
the or
equivalent of
the
of single
methane-sulfonate-treated The
experiments
the strand
ccl leave
“long
Is
open
patch”
repair
containing
DNA
made that
in
polymerase
accumulates
in
(1 I). the
question
1376
of
how
phleomycin
l-
stimulates
methyl
BIOCHEMICAL
Vol. 60, No. 4, 1974
polymerase endonuclease as
I l-induced
initiation
gests
the
dAT-rich
(7,
also
merase
is
(13,
14)
to
DNA
I I activity
of
arrest
the
of
cells
DNA
it
does
but
that
the
supplies
a to
II dAT-rich
DNA
templates
apparent
cells
these
activities
are and
(Reiter,
of
number
of
way
to
potentiate seen
triphosphates
toluenized
interacts
may
in
proteins poly-
single-stranded rapid,
in
whole
cells
that
are
available
complete
synthesis
in
acts II
on
and
III
because the
toluenized
whole
cells
activities,
they
to
(16).
regions
the
polymerase
at
molecule
enhance
phleomycin
be DNA
the
DNA-unwinding
stimulating
sug-
with
breaks
causing
that
cells It
specifically long
while
possible
not
as
phleomycin
DNA serve
specific. that
and
is
the
(12)
same
nor
unpublished),
in
containing
cells
It
toluenized
the
breaks,
relatively
regions,
whole
on
be
limited
in
anomaly in
ATP
DNA synthesis
a
RESEARCH COMMUNICATIONS
UV-induced
polypeptide
cause
unexplained.
of
template
small
at
synthesis
remains
neither
must
polymerase
bind
Finally,
in
stimulus
which
stimulates
IS),
that
ATP-independent
phleomycin
regions
which
as
for
phleomycin,
fact
breaks
sites
that
that
The
I I activity.
AND BIOPHYSICAL
do
not
have
toluenized
cells. ACKNOWLEDGMENT: Research Grant
This No.
investigation CA-17123-03Al
was from
the
supported National
by Public Cancer
Health Institute.
Service
REFERENCES
I. 2. Z:
Pisetsky, Biol. & Virasoro, Kornberg, Moses, R.
2, 5.
D.,
Berkower,
I .,
Wickner,
R.,
and
Hurwitz,
J.
(1972)
J.
Med.
557-571. and Hirota, Y. (1974) Biochimie 56, 363-371. M. L. (1971) Proc. Nat. Acad. Sci. 68, 761-764. C. C. (1971) Biochem. Biophys. Res. Commun.
Mardoh, J. and Gefter, and Richardson,
S., T. E.
1565-1971.
6. 7.
Masler, 243. Wickner, Reiter,
8.
Cozzarell
W.,
Hamewalt,
P.
W. H.,
and Komberg, Milewskiy,
M.
and
Shizuya,
A. (1973) and Kelley,
H.
(1973)
P.
Nat. (1972)
Proc.
Nature Acad. J.
New
Biol.
Sci. s, Bacterial.
2&
242-
3679-3683. fi, 586-
592. 2, 9. 10. 11. 12.
R.
and
Low,
R.
L.
(1973)
Biochem.
Biophys.
Res.
Commun.
Gass, K. B. and Cozzarelli, N. R. (1973) In Methods in Enzymology, Grossman, L. and Moldave, K., Academic Press, New York. Cooper, P. K. and Hanawalt, P. C. (1972) J. Mol. Biol. 67, l-10. Kato, K. and Strauss, 8. (1974) Proc. Nat. Acad. Sci. 71, 1969-1973. Takita, T., Mursoka, Y. and Yoshioka, T. (1972) J. Antziol. (Tokyo)
A. 5, 13. 14. 15. 16.
i , N.
151-157.
Falaschi, Pietsch, Farrell, Sigai, Proc.
eds.
Ser.
755-758. A. and Kornberg, A. (1964) Fed. P. and Garrett, H. (1968) Nature L. and Reiter, H. (1973) Antimicrob. N., Delius, H., Kornberg, T., Gefter, Nat. Acad. Sci. 69, 3537-3541.
1377
Proc. 23, 440-445. (Lond.rm, 488-489. Ag. Chemother. fr, M. L. and Alberts,
320-326. B. (1972)