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Inhibition of host cell reactivation in phage T1 by caffeine
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
INHIBITION OF HOST CELL REACTIVATION Walter Sauerbier,
IN PHAGE TI BY CAFFEINE.
Institut fHr Genetik der Universit~t
+)
zu K~in.
Received November 18, 1963
Evidence
has been compiled that host cell reactivation
(HCR) of DW irradiated phage is due to direct repair of UV lesions
(Sauerbier,
we proffered
reversion of the
in the phage and bacterial
deoxyribonucleic
(DNA). Subsequent
Howard-Flanders
b). To explain our results
of an enzymatic
support this View,
Harm,
1962a,
the hypothesis
UV photoproducts acid
1961;
experiments
by Metzger
(1963) strongly
although proof is still lacking.
et ai.,
1962; Hill,
(See also:
1958; R~rsch et ai.,
1
f9o3;
1969.)
The experiments
described
HCR can be selectively host cell reactivabie
in this paper demonstrate
inhibited by caffeine, UV photoproduct
(i) that
and (2) that the
is somehow eliminated by
HCR from the DNA of UV irradiated TI without requiring either synthesis
of Tl-messenger
ribonucieic
acid
(m-RNA)
or phage
replication. Experiments:
The UV survival
of TI is determined
after pre-
adsorption to coii B, and plating on Bs_ I (Hill, 1955), curve A). When TI is preadsorbed
in the presence
(2 gr/i00 ml), diluted through buffer containing plated on caffeine
supplemented plates
(Fig. i~
of caffeine caffeine,
and
(0.75 gr of caffeine/100
+) Dedicated to Professor Dr. Boris Rajewsky on the occasion of his 70th birthday.
340
ml
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 5E C 60
OF UV 120
180
2,;0
I\ -i
\ ~:~-2 U3
0
\
I -3"
TI 0 -4
Fi$. i
Inhibition of HCR bY caffeine. Survival of uv
irradiated TI. Curve B: with caffeine. Curve A: without caffeine. Broken line: control plating on host without HCR.
in the top agar),
curve B, Fig. i, is obtained. HCR is
abolished in more than 99% of the complexes. Direct plating of UV irradiated TI with coli B on caffeine supplemented plates leads to the same result. There is no effect of caffeine on the UV survival of TI when plated with Bs_ I.
(In these experiments
adsorption bacteria were grown in M-9 (Anderson,
341
1949) to
Vol. 14, No. 4, 1964
2 x i0 ~ ce!is/ml,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chloramphenicol
(CI~, 73 ~,/ml) was added,
and
incubation was continued for 30 minutes at 37 °. The bacteria were then shifted to TI absorption medium Benzer,
(T!-ADS; Watson,
ref.
1952 ) containing M/500 KCN. Following the CM~ treatment
all bacteria perform HCR of Ti with the same efficiency as the ~0~ fraction at direct plating of UV irradiated TI Sauerbier,
1962c).
(e.g. see
Irradiations were performed with a low
pressure mercury vapor lamp, 0SRAM HNS 12, emitting predominantly the 254 mD line with an intensity of about 3 ergs/ mm
2
see at sample distance. The second type of experiments was designed to determine
(i) whether HCR occurs in the absence of m-RNA and of DNA synthesis,
and (2) to measure the time course of HCR. As
bacterial host we used the adenine and arginine requiring coli strain B9~
(Gollub and Gots,
1959),
can be blocked by adenine withdrawal
in which RNA and DNA synthesis (Volkin, 1960). As a control
we infected washed B9~ with TI and incubated in aerated M-9 at 37 ° for 120 minutes, leased.
in which time no progeny phage was re-
(Strain B9# was kindly supplied to our lab by
Dr. E. Volkin.) Experiment: B94, arginine, in M-9
thoroughly starved for adenine and for
was infected with UV irradiated TI and incubated in
(~ 6-azauracil,
250 ~!"ml; see Pardee and Prestidge,
1960).
Then, HCR was blocked at different times after infection by plating the complexes with Bs_ I on caffeine supplemented tryptone plates. The fraction of plaque forming complexes is given in Fig. 2, curve B. In a rapidly increasing fraction of the complexes HCR becomes insensitive to caffeine. After 10-15 minutes most complexes apparently had performed HCR in the absence of m-RNA and of DNA synthesis.
(Attention is drawn to the fact that the block
342
Vol. 14, No. 4, 1964
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
of HCR must be d e l a y e d into the cells.) lengthening to about
The result
of the latent
20 minutes.
of caffeine
by the time needed is in good
period
agreement
of ~
Ti from
are plated
if HCR is not blocked
- Fig.
the caffeine
w i t h the
irradiated
W h e n the complexes
- i.e.,
to diffuse
13
on plates
2, curve
A is
obtained. h/IN 0
/NCUBATION I N M - 9 30 60 90
I -2
I
120
I
I
/
o~
o-3
-4 -O
Fi$.
2
Time
course
of DNA synthesis. times
after
control
shifted
to B94
at time
since
on caffeine
zero to M-9
2, yields
two models
Ti
and
at various
plate.
of UV irradiated
(starved
of m-RNA
plates
Curve B: caffeine
Survival
Curve B, Fig. HCR~
Plating
infection.
plate.
preadsorbed
of HCR in the absence
Curve
and arginine),
(+ 2 5 0 ~ / m i
6-azauracil)
no i n f o r m a t i o n
are compatible
343
with
A:
(150 secs),
for adenine
and at 37 ° .
on the kinetics the results.
free
of
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(1)HCR is a randomly occurring~ bacterial
instantaneous event in each
ceil. At any given time~ there are two populations
of complexes:
those in which HCR has occurred and those in
which HCR has not yet occurred. Longer times of incubation increase the probability that HCR has occurred in any given complex. (2)HCR is a gradually occurring process in every complex - i.e., there is only one population of complexes. Each reactivable UV lesion is reactivated with equal probability.
The
probability of being reactivated increases with longer times of incubation. If complete TI UV survival curves are measured with B9# treated as described above),
assumption
(pre-
(i) would predict a
series of biphasic curves when caffeine is added at dif~'erent times after infection. Assumption
(2) predicts monophasic curves
with different slopes. Fig. 3 shows that
(2) is essentially
correct. Thus, we may conclude from Fig. 2, curve B, that HCR is proceeding in every complex at approximately the same rate.
Discussion: (i) These experiments show that caffeine in concentrations of about 10-2M is an efficient and selective inhibitor of HCR in phage TI.
(2) HCR is not inhibited by caffeine,
after prolonged periods of incubation
added
(Fig. 2). This implies
that within a relatively short time HCR has achieved a state in which it is no longer sensitive to caffeine.
(3) The
occurance of HCR in the absence of m-RNA and of DNA synthesis suggests that HCR directly alters the reactivable UV photoproduct in the phage DNA. We have no detailed knowledge of the mode of caffeine action on HCR. Since photoreactivation proceeds in the presence of 3~
Vol. 14, No. 4, 1964
0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 5EC OF UV 60 90
30
120
150
@ \
~-2
\ -3
Fi$. 3
Survival curves after intermediate desrees of HCR.
UV survival of TI, preadsorbed to CMP treated,
and adenine-
arginine starved B94, plated on caffeine plates after various times of incubation in M-9
(+ 250 ~ / m l
6-azauracil)
at 37 ° .
Curve A, after 0-3 min; B, after i0 min; C, after 30 min. Curve D is plated after 40 min on plates without caffeine.
caffeine
(Metzger, 1963, personal communication),
it probably
does not form a complex with the reactivable UV photoproduct thus sterica!!y inhibiting the formation of an enzyme-substrate complex. More likely, HCR enzyme itself,
the caffeine is bound reversibly to the
since it can be readily diluted out without
affecting HCR under conditions where de novo synthesis of HCR enzyme is impossible.
345
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledsements: It is a pleasure to acknowledge the cooperation of Dr. K. Metzger and the assistance of Mrs. Gisela Goes.
REFERENCES Anderson, E.H , Proc. Natl. Aead. Sci. U.S. 32,120 (1949) Benzer, S., J. Bacteriol. 63, 59 (1952) Goilub, E.G., and Gots, J.~., J. Bacteriol. 7_~8, 320 (1959) Harm, W., Z. Vererb.-Lenre 94, 67 (1963) Hill, R.F., Biochim. Biophys. Acta 30, 636 (1958) Howard-Flanders, P., Boyce, R.P., and Theriot, L., Nature 195, 51 (1962) Metzger, K., Photoehem. Photobi01. (1963) in press; personal communication (1963) Pardee, A.B., and Prestidge, L.S., Biochim. Biophys. Aeta 37, 5#~ (196o) R~rseh, A., Edelman, A., and Cohen, J.A., Bioehim. Biophys. Acta 68, 263 ( 1 9 6 3 ) Sauerbier, W., VirologY-!5, 465 (1961); Virology 16, 398 (1962a); Z. Vererb.-Lehre 93, 220 (1962b)~-Virology 17, 164 (1962c)
3~
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
INHIBITION OF HOST CELL REACTIVATION Walter Sauerbier,
IN PHAGE TI BY CAFFEINE.
Institut fHr Genetik der Universit~t
+)
zu K~in.
Received November 18, 1963
Evidence
has been compiled that host cell reactivation
(HCR) of DW irradiated phage is due to direct repair of UV lesions
(Sauerbier,
we proffered
reversion of the
in the phage and bacterial
deoxyribonucleic
(DNA). Subsequent
Howard-Flanders
b). To explain our results
of an enzymatic
support this View,
Harm,
1962a,
the hypothesis
UV photoproducts acid
1961;
experiments
by Metzger
(1963) strongly
although proof is still lacking.
et ai.,
1962; Hill,
(See also:
1958; R~rsch et ai.,
1
f9o3;
1969.)
The experiments
described
HCR can be selectively host cell reactivabie
in this paper demonstrate
inhibited by caffeine, UV photoproduct
(i) that
and (2) that the
is somehow eliminated by
HCR from the DNA of UV irradiated TI without requiring either synthesis
of Tl-messenger
ribonucieic
acid
(m-RNA)
or phage
replication. Experiments:
The UV survival
of TI is determined
after pre-
adsorption to coii B, and plating on Bs_ I (Hill, 1955), curve A). When TI is preadsorbed
in the presence
(2 gr/i00 ml), diluted through buffer containing plated on caffeine
supplemented plates
(Fig. i~
of caffeine caffeine,
and
(0.75 gr of caffeine/100
+) Dedicated to Professor Dr. Boris Rajewsky on the occasion of his 70th birthday.
340
ml
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 5E C 60
OF UV 120
180
2,;0
I\ -i
\ ~:~-2 U3
0
\
I -3"
TI 0 -4
Fi$. i
Inhibition of HCR bY caffeine. Survival of uv
irradiated TI. Curve B: with caffeine. Curve A: without caffeine. Broken line: control plating on host without HCR.
in the top agar),
curve B, Fig. i, is obtained. HCR is
abolished in more than 99% of the complexes. Direct plating of UV irradiated TI with coli B on caffeine supplemented plates leads to the same result. There is no effect of caffeine on the UV survival of TI when plated with Bs_ I.
(In these experiments
adsorption bacteria were grown in M-9 (Anderson,
341
1949) to
Vol. 14, No. 4, 1964
2 x i0 ~ ce!is/ml,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
chloramphenicol
(CI~, 73 ~,/ml) was added,
and
incubation was continued for 30 minutes at 37 °. The bacteria were then shifted to TI absorption medium Benzer,
(T!-ADS; Watson,
ref.
1952 ) containing M/500 KCN. Following the CM~ treatment
all bacteria perform HCR of Ti with the same efficiency as the ~0~ fraction at direct plating of UV irradiated TI Sauerbier,
1962c).
(e.g. see
Irradiations were performed with a low
pressure mercury vapor lamp, 0SRAM HNS 12, emitting predominantly the 254 mD line with an intensity of about 3 ergs/ mm
2
see at sample distance. The second type of experiments was designed to determine
(i) whether HCR occurs in the absence of m-RNA and of DNA synthesis,
and (2) to measure the time course of HCR. As
bacterial host we used the adenine and arginine requiring coli strain B9~
(Gollub and Gots,
1959),
can be blocked by adenine withdrawal
in which RNA and DNA synthesis (Volkin, 1960). As a control
we infected washed B9~ with TI and incubated in aerated M-9 at 37 ° for 120 minutes, leased.
in which time no progeny phage was re-
(Strain B9# was kindly supplied to our lab by
Dr. E. Volkin.) Experiment: B94, arginine, in M-9
thoroughly starved for adenine and for
was infected with UV irradiated TI and incubated in
(~ 6-azauracil,
250 ~!"ml; see Pardee and Prestidge,
1960).
Then, HCR was blocked at different times after infection by plating the complexes with Bs_ I on caffeine supplemented tryptone plates. The fraction of plaque forming complexes is given in Fig. 2, curve B. In a rapidly increasing fraction of the complexes HCR becomes insensitive to caffeine. After 10-15 minutes most complexes apparently had performed HCR in the absence of m-RNA and of DNA synthesis.
(Attention is drawn to the fact that the block
342
Vol. 14, No. 4, 1964
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
of HCR must be d e l a y e d into the cells.) lengthening to about
The result
of the latent
20 minutes.
of caffeine
by the time needed is in good
period
agreement
of ~
Ti from
are plated
if HCR is not blocked
- Fig.
the caffeine
w i t h the
irradiated
W h e n the complexes
- i.e.,
to diffuse
13
on plates
2, curve
A is
obtained. h/IN 0
/NCUBATION I N M - 9 30 60 90
I -2
I
120
I
I
/
o~
o-3
-4 -O
Fi$.
2
Time
course
of DNA synthesis. times
after
control
shifted
to B94
at time
since
on caffeine
zero to M-9
2, yields
two models
Ti
and
at various
plate.
of UV irradiated
(starved
of m-RNA
plates
Curve B: caffeine
Survival
Curve B, Fig. HCR~
Plating
infection.
plate.
preadsorbed
of HCR in the absence
Curve
and arginine),
(+ 2 5 0 ~ / m i
6-azauracil)
no i n f o r m a t i o n
are compatible
343
with
A:
(150 secs),
for adenine
and at 37 ° .
on the kinetics the results.
free
of
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(1)HCR is a randomly occurring~ bacterial
instantaneous event in each
ceil. At any given time~ there are two populations
of complexes:
those in which HCR has occurred and those in
which HCR has not yet occurred. Longer times of incubation increase the probability that HCR has occurred in any given complex. (2)HCR is a gradually occurring process in every complex - i.e., there is only one population of complexes. Each reactivable UV lesion is reactivated with equal probability.
The
probability of being reactivated increases with longer times of incubation. If complete TI UV survival curves are measured with B9# treated as described above),
assumption
(pre-
(i) would predict a
series of biphasic curves when caffeine is added at dif~'erent times after infection. Assumption
(2) predicts monophasic curves
with different slopes. Fig. 3 shows that
(2) is essentially
correct. Thus, we may conclude from Fig. 2, curve B, that HCR is proceeding in every complex at approximately the same rate.
Discussion: (i) These experiments show that caffeine in concentrations of about 10-2M is an efficient and selective inhibitor of HCR in phage TI.
(2) HCR is not inhibited by caffeine,
after prolonged periods of incubation
added
(Fig. 2). This implies
that within a relatively short time HCR has achieved a state in which it is no longer sensitive to caffeine.
(3) The
occurance of HCR in the absence of m-RNA and of DNA synthesis suggests that HCR directly alters the reactivable UV photoproduct in the phage DNA. We have no detailed knowledge of the mode of caffeine action on HCR. Since photoreactivation proceeds in the presence of 3~
Vol. 14, No. 4, 1964
0
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 5EC OF UV 60 90
30
120
150
@ \
~-2
\ -3
Fi$. 3
Survival curves after intermediate desrees of HCR.
UV survival of TI, preadsorbed to CMP treated,
and adenine-
arginine starved B94, plated on caffeine plates after various times of incubation in M-9
(+ 250 ~ / m l
6-azauracil)
at 37 ° .
Curve A, after 0-3 min; B, after i0 min; C, after 30 min. Curve D is plated after 40 min on plates without caffeine.
caffeine
(Metzger, 1963, personal communication),
it probably
does not form a complex with the reactivable UV photoproduct thus sterica!!y inhibiting the formation of an enzyme-substrate complex. More likely, HCR enzyme itself,
the caffeine is bound reversibly to the
since it can be readily diluted out without
affecting HCR under conditions where de novo synthesis of HCR enzyme is impossible.
345
Vol. 14, No. 4, 1964
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledsements: It is a pleasure to acknowledge the cooperation of Dr. K. Metzger and the assistance of Mrs. Gisela Goes.
REFERENCES Anderson, E.H , Proc. Natl. Aead. Sci. U.S. 32,120 (1949) Benzer, S., J. Bacteriol. 63, 59 (1952) Goilub, E.G., and Gots, J.~., J. Bacteriol. 7_~8, 320 (1959) Harm, W., Z. Vererb.-Lenre 94, 67 (1963) Hill, R.F., Biochim. Biophys. Acta 30, 636 (1958) Howard-Flanders, P., Boyce, R.P., and Theriot, L., Nature 195, 51 (1962) Metzger, K., Photoehem. Photobi01. (1963) in press; personal communication (1963) Pardee, A.B., and Prestidge, L.S., Biochim. Biophys. Aeta 37, 5#~ (196o) R~rseh, A., Edelman, A., and Cohen, J.A., Bioehim. Biophys. Acta 68, 263 ( 1 9 6 3 ) Sauerbier, W., VirologY-!5, 465 (1961); Virology 16, 398 (1962a); Z. Vererb.-Lehre 93, 220 (1962b)~-Virology 17, 164 (1962c)
3~