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The biosynthesis of a pyrimidine replacing thymine in bacteriophage DNA
Vol. 16, No. 2, 1964
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
THE BIOSYNTHEEIS OF A PYHIMIDINE REPLACING THYMINE IN BACTERIOPHAGE DNA D. H. Roscoe* and R. G. Tucker Microbiology
Unit,
Department
of Biochemistry,
University
of
Oxford Received
April
20,
1964
A phage, de, which attacks subtilis,
a Marburg strain
NCTC 3610, has been isolated
composition present
determined.
Adenine,
from soil
and its
guanine and cytosine
(G+C = 40.3%) but thymine is completely
5-hydroxymethyl
uracil.
of de DNA gives
a Tm of 76.50 in 1.5 x 10"
&sodium tilis
of Bacillus
citrate.
phage, SP-8, analysed
by Kallen,
There are however some differences rde has a head diameter while
respectively
&NaCl
(Davison,
values
length
of 70 rnp
also no glucose has been detected and Marmur, 1963).
on infection
been studied
that
indicate
by de have
5-hydroxymethyl-
acid (dHMUMP) is formed from deoxyuridylic
(dUMPI by a hydroxymethylation
reaction
methyldeoxyeytidylate-synthesising infeoted
Escherichia
coli
*Medical
Research Council
sub-
between the two phagesr
Some of the enzymic changes occurring deoxyuridylic
+ 1.5 x 10m2
to another
to SP-8 (Takahashi
and the results
transition
for SP-8 are 100 rnp and 170 rnlr
1963);
bound to de DNA in contrast
by
Simon and Marmur (1962).
of 50 rnp and a tail
the corresponding
are
replaced
Measurement of the thermal
The phage is thus similar
DNA
analogous
acid
to the hydroxy-
mechanism found in phage-
(Flaks and Cohen, 19591. Grantee 106
Vol. 16, No. 2, 1964
BIOCHEMICAL
(i)
Methods.
Crude extracts
grown with vigorous medium (Davis
AND BIOPHYSICAL
aeration
were prepared
at 37” in double
and Mingioli,
1950) containing
Organisms in the log phase of growth infected
with
a five-fold
tinued
for
disrupted
and other
at 10,000 g for
and aeration
were
buffer,
Ultrasonic particles
con-
chilled
by centrifugation.
to 2O
After
washing
pH 7.5, the cells Generator,
were
Type E 7590B.
were removed by centrifugatlon
5 min. and 100,000 g for
bacteria
minimal
4 x 10-a &glucose.
were rapidly
in 10-l &Tris
using a Mullard
Unbroken cells uninfected
The cultures collected
and resuspension
strength
excess of de (which has a latent
of 60 min. under these conditions)
and the organisms
from cultures
(5 x lo* cells/ml.)
period
20 min.
RESEARCH COMMUNICATIONS
were prepared
60 min.
Extracts
from
by the same method from parallel
cultures. (ii)
Deoxycytidyllc
photometrically 175 moles
pH 7.5,
(1962). 50 moles
tetrahydrofollc
acid
(THFA)
volume of 0.45 ml.
To start
added to one cuvette followed at 250.
and the increase
and Discussion.
contained:
mercaptoethanol,
0.1 moles
in Table 1.
according
25 wales
12.5 Crmoles MgCls,
10 poles
formaldehyde,
and extract
in a total
the reaction
0.05 moles
in optical
dUMF'were
density
at 340 m)L
The changes in the specific
of dCMP deaminase and TMP synthetase
oucurs during
pre-
of decrease
was estimated
Cuvettes
ethylenediaminetetraacetate,
are given
dCMP; freshly
and the rate
(TMP) synthetase
0.5 moles
activities
in 3.5 ml:
at 285 rnp measured at 250.
to Wahba and Friedkin
Results
contained
pH 7.5, and 1 mole
Thymidylate
Tris buffer,
Each cuvette
was added to one cuvette
density
(iii?
(dCMP) deaminase was measured spectro-
(Wang, 1955).
Tris buffer,
pared extract in optical
acid
An increase
on infection
in dCMP deaminase also
growth of SP-8 (Marmur and Greenspan, 107
1963) and
Vol. 16, No. 2, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Comparison of dCMP deaminase and TMB synthetase activities in extracts of B. subtilis, uninfected and infected with de. Extract
*dCMP deaminase
Uninfected
*TMB synthetase
< 0.1 2.0
Infected
0.015 < 0.001
*Units of activity: dCMB deaminase, wales dCMB utilised/mg. protein/hr.; TMP synthetase , wales THFA oxidised/mg. protein /hr.
this
change suggests that
dUMP, is an intermediate of Neuroscora (Abbott,
convert
activity
thymine and thymidine
hydroxymethylation
this
reaction
it
system.
pH 7.5,
1 wale
dUMP in a total
pathway is about
in boiling
protein
The supernatant
removed.
No. 1 chromatography n-butanol/ammonia
and the resulting
water for fluid
THFA and
The reaction
was
5 min. and precipitated was applied
to Whatman
paper and the spots washed overnight
with
(Markham and Smith, 1949) to remove unwanted
material.
were eluted
mercaptoethanol,
0.2 mole
volume of 1 ml.
stopped by heating
The nucleotides, with water , treated
nucleosides
which remain at the with alkaline
paper-chromatographed
(Markham and Smith, 1949).
was detected
that a similar
for 45 min. at 37* with
10 poles
H'*CHO (1.1 x lo6 cpm/mole),
n-butanol
seems unlikely
of dUMP and an enzyme bringing
1 wale
origin,
to HMU derivatives
A more probable
were incubated
Tris buffer,
radioactive
Extracts
has been detected.
Enzymic extracts 50 Poles
of dHMUMP.
1964) but with the decrease of TMB
on infection
step occurs in the viral direct
of the enzymic reaction,
in the biosynthesis
Kadner and Fink,
synthetase
the product
and had the chromatographic 108
phosphatase in 86% aq.
A peak of radioactivity properties
of synthetic
BIOCHEMICAL
Vol. 16, No. 2, 1964
HMU deoxyriboside
in
/ammonia
(Markham
and Smith,
1951).
The radioactive
Control
are
/hr.)
ment
is
not
infected but
2. means
n-butanol
and n-butanol
and isopropanol/HCl
was eluted
of the
in
with
the
sufficient
moles
possibility
and the The results
eluted.
activity
dHMUMP formed/mg.
to account This
and counted.
same manner
chromatogram
(0.01
(Wyatt,
water
The dUMP hydroxymethylase
bacteria. the
1949)
were treated
Table
by this
86% aq.
area
regions
shown in
detected
in
3 solvents:
incubations
corresponding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
the
rate
may be due to the
of a second
pathway
protein
of DNA synthesis method
cannot
of measurebe eliminated.
TABLE 2 Fixation B. subtilis,
of HK4CH0 into
Incubation *Complete
1, II
11 II
**Reagent
mixture
I!
rate
5600 minus II ,l II
dUMP
125
THFA
65
mercaptoethanol
4450
extract
+ boiled
II
95
extract
+ extract
105
of uninfected
cells
system as described in text from infected bacteria was heated water
has been
of synthesis
1959 a);
it
methylase
could
suggest
mixture
system 11 I,
It
of be-infected cpm/ml.
mixture
II II
*Complete **Extract boiling
dHMUMP by extracts
that
is
the
suggested
that
not exert
thymine
known at this a similar
dCMP deaminase
for
TMP synthetase
of DNA containing
stage
115 5 min.
limits (Flaks
whether
the and Cohen,
the hydroxy-
control.
Preliminary
is
to inhibition
109
subject
in
results by
Vol. 16, No. 2, 1964
BIOCHEMICAL
dUMP and TMP, and in this found in rapidly
growing
de Petrocellis,
1960;
of TMP synthetase production Friedkin,
the synthetase in de-infected
(Scarano,
similar
it
of the latter
The reduction
(1962);
if
by
extract
from normal bacteria
is decreased,
appears that dIiMUMP can
B, subtilis
Bonaduce and
to that demonstrated
is mixed with that
activity
that
may be caused by the increased
Donovan and Pastore
bacteria
In conclusion
the enzyme is like
Maley and Maley, 1962).
of an inhibitor
from infected
respeot
animal tissue
activity
Crawford,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by the following
be
synthesised
pathway:
dUMP hydroxymethylase I I L. - !i%Ge,d l TMp Acknowledaementsr We are indebted to Dr D. Kay for electron micrographs and to the British Empire Cancer Campaign for financial help.
References Abbot&MZjb
Kadner,
R.J. & Fink,
R.M. (19641,
J. biol.
Chem.
Davis, B:D. &*Min ioli, E.S. (1950>, J. Bact. 60, 17. Davison, P.F. (19 %3), Virology, 2, 146, Flaks, J.G. & Cohen, S.S. (1959), J. biol. Chem. a, 1501. Flaks, J.G. & Cohen, S.S. (1959 a), J. biol. Chem. 214, 2981. Friedkin M., Crawford, E.J., Donovan, E. & Pastore, E.J. (19b2), J. biol. Chem. m, 3811. Kallen, R.G., Simon, M. & Marmur, J. (1962!, J. molec. Biol. 5, 248. Maley, G.F. & Maley, F. (1962), J. biol. Markham, R. & Smith, J.D. (1949) Marmur, J. & Greenspan, Scarano, E., Bonaduce, L, & de Petrocellis, J. biol. Chem. m, 3556. Takahashi, I. & Marmur, J. (1963), Biochem. Biophys. Res. CODUU. 10, 289. Wahba, A.J. & Friedkin, M. (1962), J. biol. Chem. u, 3794. Wang, T.P. (19551, in Methods in Enzymology, vol II, p. 478. Ed. Colowick, S.P. & Kaplan, N.O., New York, Academic Press, Wyatt, G.R. (1951), Biochem. J. 48, 584. 110
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
THE BIOSYNTHEEIS OF A PYHIMIDINE REPLACING THYMINE IN BACTERIOPHAGE DNA D. H. Roscoe* and R. G. Tucker Microbiology
Unit,
Department
of Biochemistry,
University
of
Oxford Received
April
20,
1964
A phage, de, which attacks subtilis,
a Marburg strain
NCTC 3610, has been isolated
composition present
determined.
Adenine,
from soil
and its
guanine and cytosine
(G+C = 40.3%) but thymine is completely
5-hydroxymethyl
uracil.
of de DNA gives
a Tm of 76.50 in 1.5 x 10"
&sodium tilis
of Bacillus
citrate.
phage, SP-8, analysed
by Kallen,
There are however some differences rde has a head diameter while
respectively
&NaCl
(Davison,
values
length
of 70 rnp
also no glucose has been detected and Marmur, 1963).
on infection
been studied
that
indicate
by de have
5-hydroxymethyl-
acid (dHMUMP) is formed from deoxyuridylic
(dUMPI by a hydroxymethylation
reaction
methyldeoxyeytidylate-synthesising infeoted
Escherichia
coli
*Medical
Research Council
sub-
between the two phagesr
Some of the enzymic changes occurring deoxyuridylic
+ 1.5 x 10m2
to another
to SP-8 (Takahashi
and the results
transition
for SP-8 are 100 rnp and 170 rnlr
1963);
bound to de DNA in contrast
by
Simon and Marmur (1962).
of 50 rnp and a tail
the corresponding
are
replaced
Measurement of the thermal
The phage is thus similar
DNA
analogous
acid
to the hydroxy-
mechanism found in phage-
(Flaks and Cohen, 19591. Grantee 106
Vol. 16, No. 2, 1964
BIOCHEMICAL
(i)
Methods.
Crude extracts
grown with vigorous medium (Davis
AND BIOPHYSICAL
aeration
were prepared
at 37” in double
and Mingioli,
1950) containing
Organisms in the log phase of growth infected
with
a five-fold
tinued
for
disrupted
and other
at 10,000 g for
and aeration
were
buffer,
Ultrasonic particles
con-
chilled
by centrifugation.
to 2O
After
washing
pH 7.5, the cells Generator,
were
Type E 7590B.
were removed by centrifugatlon
5 min. and 100,000 g for
bacteria
minimal
4 x 10-a &glucose.
were rapidly
in 10-l &Tris
using a Mullard
Unbroken cells uninfected
The cultures collected
and resuspension
strength
excess of de (which has a latent
of 60 min. under these conditions)
and the organisms
from cultures
(5 x lo* cells/ml.)
period
20 min.
RESEARCH COMMUNICATIONS
were prepared
60 min.
Extracts
from
by the same method from parallel
cultures. (ii)
Deoxycytidyllc
photometrically 175 moles
pH 7.5,
(1962). 50 moles
tetrahydrofollc
acid
(THFA)
volume of 0.45 ml.
To start
added to one cuvette followed at 250.
and the increase
and Discussion.
contained:
mercaptoethanol,
0.1 moles
in Table 1.
according
25 wales
12.5 Crmoles MgCls,
10 poles
formaldehyde,
and extract
in a total
the reaction
0.05 moles
in optical
dUMF'were
density
at 340 m)L
The changes in the specific
of dCMP deaminase and TMP synthetase
oucurs during
pre-
of decrease
was estimated
Cuvettes
ethylenediaminetetraacetate,
are given
dCMP; freshly
and the rate
(TMP) synthetase
0.5 moles
activities
in 3.5 ml:
at 285 rnp measured at 250.
to Wahba and Friedkin
Results
contained
pH 7.5, and 1 mole
Thymidylate
Tris buffer,
Each cuvette
was added to one cuvette
density
(iii?
(dCMP) deaminase was measured spectro-
(Wang, 1955).
Tris buffer,
pared extract in optical
acid
An increase
on infection
in dCMP deaminase also
growth of SP-8 (Marmur and Greenspan, 107
1963) and
Vol. 16, No. 2, 1964
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Comparison of dCMP deaminase and TMB synthetase activities in extracts of B. subtilis, uninfected and infected with de. Extract
*dCMP deaminase
Uninfected
*TMB synthetase
< 0.1 2.0
Infected
0.015 < 0.001
*Units of activity: dCMB deaminase, wales dCMB utilised/mg. protein/hr.; TMP synthetase , wales THFA oxidised/mg. protein /hr.
this
change suggests that
dUMP, is an intermediate of Neuroscora (Abbott,
convert
activity
thymine and thymidine
hydroxymethylation
this
reaction
it
system.
pH 7.5,
1 wale
dUMP in a total
pathway is about
in boiling
protein
The supernatant
removed.
No. 1 chromatography n-butanol/ammonia
and the resulting
water for fluid
THFA and
The reaction
was
5 min. and precipitated was applied
to Whatman
paper and the spots washed overnight
with
(Markham and Smith, 1949) to remove unwanted
material.
were eluted
mercaptoethanol,
0.2 mole
volume of 1 ml.
stopped by heating
The nucleotides, with water , treated
nucleosides
which remain at the with alkaline
paper-chromatographed
(Markham and Smith, 1949).
was detected
that a similar
for 45 min. at 37* with
10 poles
H'*CHO (1.1 x lo6 cpm/mole),
n-butanol
seems unlikely
of dUMP and an enzyme bringing
1 wale
origin,
to HMU derivatives
A more probable
were incubated
Tris buffer,
radioactive
Extracts
has been detected.
Enzymic extracts 50 Poles
of dHMUMP.
1964) but with the decrease of TMB
on infection
step occurs in the viral direct
of the enzymic reaction,
in the biosynthesis
Kadner and Fink,
synthetase
the product
and had the chromatographic 108
phosphatase in 86% aq.
A peak of radioactivity properties
of synthetic
BIOCHEMICAL
Vol. 16, No. 2, 1964
HMU deoxyriboside
in
/ammonia
(Markham
and Smith,
1951).
The radioactive
Control
are
/hr.)
ment
is
not
infected but
2. means
n-butanol
and n-butanol
and isopropanol/HCl
was eluted
of the
in
with
the
sufficient
moles
possibility
and the The results
eluted.
activity
dHMUMP formed/mg.
to account This
and counted.
same manner
chromatogram
(0.01
(Wyatt,
water
The dUMP hydroxymethylase
bacteria. the
1949)
were treated
Table
by this
86% aq.
area
regions
shown in
detected
in
3 solvents:
incubations
corresponding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
the
rate
may be due to the
of a second
pathway
protein
of DNA synthesis method
cannot
of measurebe eliminated.
TABLE 2 Fixation B. subtilis,
of HK4CH0 into
Incubation *Complete
1, II
11 II
**Reagent
mixture
I!
rate
5600 minus II ,l II
dUMP
125
THFA
65
mercaptoethanol
4450
extract
+ boiled
II
95
extract
+ extract
105
of uninfected
cells
system as described in text from infected bacteria was heated water
has been
of synthesis
1959 a);
it
methylase
could
suggest
mixture
system 11 I,
It
of be-infected cpm/ml.
mixture
II II
*Complete **Extract boiling
dHMUMP by extracts
that
is
the
suggested
that
not exert
thymine
known at this a similar
dCMP deaminase
for
TMP synthetase
of DNA containing
stage
115 5 min.
limits (Flaks
whether
the and Cohen,
the hydroxy-
control.
Preliminary
is
to inhibition
109
subject
in
results by
Vol. 16, No. 2, 1964
BIOCHEMICAL
dUMP and TMP, and in this found in rapidly
growing
de Petrocellis,
1960;
of TMP synthetase production Friedkin,
the synthetase in de-infected
(Scarano,
similar
it
of the latter
The reduction
(1962);
if
by
extract
from normal bacteria
is decreased,
appears that dIiMUMP can
B, subtilis
Bonaduce and
to that demonstrated
is mixed with that
activity
that
may be caused by the increased
Donovan and Pastore
bacteria
In conclusion
the enzyme is like
Maley and Maley, 1962).
of an inhibitor
from infected
respeot
animal tissue
activity
Crawford,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
by the following
be
synthesised
pathway:
dUMP hydroxymethylase I I L. - !i%Ge,d l TMp Acknowledaementsr We are indebted to Dr D. Kay for electron micrographs and to the British Empire Cancer Campaign for financial help.
References Abbot&MZjb
Kadner,
R.J. & Fink,
R.M. (19641,
J. biol.
Chem.
Davis, B:D. &*Min ioli, E.S. (1950>, J. Bact. 60, 17. Davison, P.F. (19 %3), Virology, 2, 146, Flaks, J.G. & Cohen, S.S. (1959), J. biol. Chem. a, 1501. Flaks, J.G. & Cohen, S.S. (1959 a), J. biol. Chem. 214, 2981. Friedkin M., Crawford, E.J., Donovan, E. & Pastore, E.J. (19b2), J. biol. Chem. m, 3811. Kallen, R.G., Simon, M. & Marmur, J. (1962!, J. molec. Biol. 5, 248. Maley, G.F. & Maley, F. (1962), J. biol. Markham, R. & Smith, J.D. (1949) Marmur, J. & Greenspan, Scarano, E., Bonaduce, L, & de Petrocellis, J. biol. Chem. m, 3556. Takahashi, I. & Marmur, J. (1963), Biochem. Biophys. Res. CODUU. 10, 289. Wahba, A.J. & Friedkin, M. (1962), J. biol. Chem. u, 3794. Wang, T.P. (19551, in Methods in Enzymology, vol II, p. 478. Ed. Colowick, S.P. & Kaplan, N.O., New York, Academic Press, Wyatt, G.R. (1951), Biochem. J. 48, 584. 110