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Degradation of thymus DNA and crab poly d(A-T) by micrococcal nuclease in the presence of actinomycin D
207
SHORT COMMUNICATIONS
BBA 93299
Degradation of thymus DNA and crab poly d(A-T) by micrococcal nuclease in the presence of actinomycin D The present communication deals with the "shielding" effect of actinomycin D on DNA and crab poly d(A-T) against the action of micrococcal nuclease (EC 3.1.4.7). Two substrates with different binding capacities for actinomycin D were used: thymus DNA and crab p~ly d(A-T) 1. Thymus DNA binds about I molecule of actinomycin D per 7 base pairs 2,3, whereas crab poly d(A-T) binds I per 56 base pairs ~. Several authors have studied the effect of actinomycin D on the degradation of DNA b y different nucleases and arrived at conflicting conclusions4-~. Recently SARKAR7 used very small amounts of pancreatic deoxyribonuclease r, spleenic deoxyribonuclease Ir and venom phosphodiesterase (exonuclease) which allowed him to study the early stages of degradation of DNA. He observed that actinomycin D inhibited the action of all three enzymes by about 70%. rn our experiments large amounts of micrococcal nuclease 8 (virtually phosphatase-free) were used to carry the reaction to completion; this permitted an evaluation of the effect of actinomycin D at different stages of the reaction. Since the effect varied with the stage of reaction, the previously observed discrepancies may now be resolved. Hydrolysis was followed using the pH-stat described by PEANASKYAND SZUCS9. The enzymatic reactions were carried out at p H 9.0 in the presence of 2. 5 mM CaC12 using o.oi M NaOH as titrant. A steady stream of water saturated nitrogen was passed over the incubation mixtttre kept in a water jacketed cell at 37 °. The NaOH storage and delivery systems were as described by RICHARDSet al. 1°. Crystalline, phosphatasefree micrococcal nuclease was prepared as described previously 8. Thymus DNA was prepared according to KAY, SIMMONS AND DOUNCE 11, and crab poly d(A-T)prepared from testes of Cancer borealis (B. BARANOWSKA, T. BARANOWSKI AND H. LASKOWSKI, Sr.)12 was a gift of Professor and Mrs. T. BARANOWSKI. Actinomycin D was a gift of Merck, Sharp, and Dohme. Crystallized bovine albumin was obtained from Pentex. Fig. IA illustrates the effect of actinomycin D on the degradation of thymus DNA. During early stages of the reaction a strong inhibition of at least 70% of the control was observed. As the reaction progressed there was a continuous decrease of the inhibition by actinomycin D. Chromatography of the complete digests obtained in the presence or absence of actinomycin D showed mononucleotides and dinucleotides in a molar ratio of 2 : i, indicating that the extent of hydrolysis is independent of actinomycin D. Only traces of trinucleotides were detectable. Since actinomycin D is unstable in strongly alkaline solutions (Dr. J. HURWlTZ, personal communication) a mixture of thymus DNA and actinomycin D was preincubated at pH 9.0 for 60 rain prior to the addition of enzyme. Fig. IB shows that effect of actinomycin D increased rather than decreased, presumably because binding to DNA is a time-dependent reaction. Fig. 2 shows that the inhibitory effect of actinomycin D on the degradation of crab poly d (A-T) was small. The inhibition was undetectable during early stages Biochim. Biophys. Acta, 157 (1968) 2o7-2o9
208
SHORT COMMUNICATIONS
2.C
1.5
~
-r 0 o z
tt ] .0
i"
1.5
~'~
-g
~
~,,/
1.0
o
z
g 0,5
=k 0 . 5
S~/t
:a
B
A o
(o
20
~'o ,'o
5~ 6o
,b
/0
3o
TIME (rain)
TIME (rain)
Fig. i. H y d r o l y s i s of t h y m u s DNA. A. , 15 AZ00m# u n i t s of D N A in 4 ml of 2. 5 mM CaCI, were i n c u b a t e d for 20 m i n a t 37 °. The p H w a s a d j u s t e d to 9.0, and the base line of N a O H u p t a k e w a s r u n for io min. The reaction w a s s t a r t e d b y the addition of o.i u n i t of micrococcal nuctease in 20/zl of o . 1 % s e r u m albumin. - - - , 15 A260m/* u n i t s of D N A in 4 ml of 2.5 mM CaCI= were i n c u b a t e d for zo min at 37 °. The p H w a s a d j u s t e d to 9.o, a n d the base line of N a O H u p t a k e w a s r u n for io min. A c t i n o m y c i n D (ioo/~g) w a s added, t h e p H r e a d j u s t e d to 9.o, and the reaction w a s s t a r t e d w i t h i n 1-2 m i n b y the a d d i t i o n of o. i u n i t of micrococcal nuclease in 2o/zl of s e r u m albumin. The procedure described in A w a s followed r o u t i n e l y in all o t h e r experiments, unless otherwise stated. B. , 15 A260m# u n i t s of DXA, 2.5 mM CaCI,, 0.2 u n i t of enzyme. Detailed procedure as in A. - - -, 15 A260m/* u n i t s of DNA, 2. 5 mM CaCI=, IOO/*g of a c t i n o m y c i n D, 0.2 u n i t of enzyme. Detailed p r o c e d u r e as in A. { } - 0 , 15 A260m# u n i t s of DNA, 2,5 mM CaC1v A c t i n o m y c i n D w a s added, the p H r e a d j u s t e d to 9.o, and t h e m i x t u r e w a s p r e i n c u b a t e d for 60 min. The reaction w a s s t a r t e d w i t h o.2 u n i t of enzyme. O t h e r details as described in A.
of hydrolysis. As the reaction proceeded, the A-T regions of the substrate were hydrolyzed preferentially and as a result the G-C regions were relatively increased. Under such conditions the inhibition by actinomycin D was detectable. During the final stages of the reaction the inhibition again disappeared. The exhaustive digests
2.0
2.0 :a:: o :~o
"TO
7°
1.0
o E :3, 0.5
o
P 0.5 :a,
o
1.0
to 2o TIME (rain)
=,o
0
1'0 20 TIME (rnin)
50
Fig. 2. H y d r o l y s i s of crab d ( A - T ) . - , 15 A260m# u n i t s of crab poly d ( A - T ) , 2.5 mM CaCI~, o,o6 u n i t of e n z y m e . Details of p r o c e d u r e as described in Fig. iA. - - - , 15 A260m# units of crab poly d ( A - T ) , 2.5 mM CaC1 v IOO/,g of a c t i n o m y c i n D, 0.o6 u n i t of enzyme. Detailed procedure as in Fig. iA. Fig. 3. H y d r o l y s i s of a m i x t u r e of c r a b p o l y d ( A - T ) (95~o) a n d t h y m u s D N A (5%). - - , I5 A260m# u n i t s of the m i x t u r e , 2.5 mM CaC12, 0.08 u n i t of enzyme. Detailed procedure as in Fig. iA. - - - , 15 A260 m/* u n i t s of the m i x t u r e , 2.5 mM CaCIz, i o o / z g of a c t i n o m y c i n D, o,08 u n i t of enzyme. Detailed procedure as in Fig. iA.
Biochim. Biophys. Acta, 157 (1968) 2o7-2o9
209
SHORT COMMUNICATIONS
contained only mononucleotides and dinucleotides in a molar ratio of 2:1. This is typical for all digests of crab poly d(A-T) by micrococcal nuclease. Fig. 3 illustrates experiments in which crab poly d(A-T) (95~) was mixed with DNA (5%). Tile inhibition by actinomycin D was greater in the presence of both substrates than when crab poly d(A-T) was the sole substrate. It seems possible that differences in susceptibility to inhibition by actinomycin D can serve as an analytical test for impurities in crab poly d(A-T) preparations. In conclusion, the "shielding" by actinomycin D of polynucleotides against degradation by micrococcal nuclease depends on the nature of the substrate and on the stage of the reaction at which the effect is measured. It seems possible that actinomycin D could be used in those cases in which the protection of G-C-containing oligonucleotide fragments from further degradation is desired. This investigation was supported by Grants E-I571 and PRP-3o from the American Cancer Society, GB-6o58 from the National Science Foundation and by Contract AT(3o-I)363o from the Atomic Energy Commission.
Laboratory o/ Enzymology, Roswell Park Memorial Institute, Bu//alo, N. Y. 142o3 (U.S.A.)
E. SULKOWSKI M. LASKOWSKI, SR.
I. H. GOLDBERG, 1VI. RABINOWITZ AND E. REICH, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2094. M. GELLERT, C. E. SMITH, D. NEVILLE AND G. FELSENFELD, J. Mol. Biol., l i (1965) 445. R. W. HYMAN AND •. DAVIDSON, Biochem. Biophys. Res. Commun., 26 (1967) 116. J. D. LEITH, Biochim. Biophys. Acta., 72 (1963) 643. B. R. McAIJSLAN, Biochem. Biophys. Res. Commun., 19 (1965) 15. L. J. ERON AND B. R. McAusLAN, Biochim. Biophys. Acta, 114 (1966) 633. N. K. SARKAR,Biochim. Biophys. Acta, I45 (1967) 174. E. SULKOWSKI AND M. LASKOWSKI, SR., J. Biol. Chem., 241 (1966) 4386. R. J. PEANASKY AND M. S z u c s , J. Biol. Chem., 239 (1964) 2525. G. M. RICHARDS, D. J. TUTAS, W. J. WECHTER AND M. LASKOWSKI, SR., Biochemistry, 6 (1967) 29o8. i i E. R. M. KAY, N. S. SIMMONS AND A. L. DOUNCE, J. Am. Chem. Soc., 74 (1952) 1724. I2 ]3. BARANOWSKI, T. BARANOWSKI, H. LASKOWSKI, Sr., European J. Biochemistry, (1968) in t h e press. I 2 3 4 5 6 7 8 9 io
Received December Ilth, 1967 Biochim. Biophys. Acta, 157 (1968) 2o7-2o 9 BBA 93297
Isolation and properties of D N A from eggs and gastrulae of Ascaris lurnbricoides In experiments on regulation processes of transcription during the early mosaic development of Ascaris, we studied the conditions for isolation of high molecular weight DNA, and some properties of the DNA preparations. The results of these experiments are described in this paper. The isolation of native DNA in substantial amounts can be achieved by a combined sodium dodecyl sulfate-chloroform-phenol procedure. The eggs or gastrulae were suspended in 0.2 M NaCl-o.I M EDTA-o.oI M Tris-HC1 solution (pH8) Biochim. Biophys. Acta, i57 (1968) 2o9-212
SHORT COMMUNICATIONS
BBA 93299
Degradation of thymus DNA and crab poly d(A-T) by micrococcal nuclease in the presence of actinomycin D The present communication deals with the "shielding" effect of actinomycin D on DNA and crab poly d(A-T) against the action of micrococcal nuclease (EC 3.1.4.7). Two substrates with different binding capacities for actinomycin D were used: thymus DNA and crab p~ly d(A-T) 1. Thymus DNA binds about I molecule of actinomycin D per 7 base pairs 2,3, whereas crab poly d(A-T) binds I per 56 base pairs ~. Several authors have studied the effect of actinomycin D on the degradation of DNA b y different nucleases and arrived at conflicting conclusions4-~. Recently SARKAR7 used very small amounts of pancreatic deoxyribonuclease r, spleenic deoxyribonuclease Ir and venom phosphodiesterase (exonuclease) which allowed him to study the early stages of degradation of DNA. He observed that actinomycin D inhibited the action of all three enzymes by about 70%. rn our experiments large amounts of micrococcal nuclease 8 (virtually phosphatase-free) were used to carry the reaction to completion; this permitted an evaluation of the effect of actinomycin D at different stages of the reaction. Since the effect varied with the stage of reaction, the previously observed discrepancies may now be resolved. Hydrolysis was followed using the pH-stat described by PEANASKYAND SZUCS9. The enzymatic reactions were carried out at p H 9.0 in the presence of 2. 5 mM CaC12 using o.oi M NaOH as titrant. A steady stream of water saturated nitrogen was passed over the incubation mixtttre kept in a water jacketed cell at 37 °. The NaOH storage and delivery systems were as described by RICHARDSet al. 1°. Crystalline, phosphatasefree micrococcal nuclease was prepared as described previously 8. Thymus DNA was prepared according to KAY, SIMMONS AND DOUNCE 11, and crab poly d(A-T)prepared from testes of Cancer borealis (B. BARANOWSKA, T. BARANOWSKI AND H. LASKOWSKI, Sr.)12 was a gift of Professor and Mrs. T. BARANOWSKI. Actinomycin D was a gift of Merck, Sharp, and Dohme. Crystallized bovine albumin was obtained from Pentex. Fig. IA illustrates the effect of actinomycin D on the degradation of thymus DNA. During early stages of the reaction a strong inhibition of at least 70% of the control was observed. As the reaction progressed there was a continuous decrease of the inhibition by actinomycin D. Chromatography of the complete digests obtained in the presence or absence of actinomycin D showed mononucleotides and dinucleotides in a molar ratio of 2 : i, indicating that the extent of hydrolysis is independent of actinomycin D. Only traces of trinucleotides were detectable. Since actinomycin D is unstable in strongly alkaline solutions (Dr. J. HURWlTZ, personal communication) a mixture of thymus DNA and actinomycin D was preincubated at pH 9.0 for 60 rain prior to the addition of enzyme. Fig. IB shows that effect of actinomycin D increased rather than decreased, presumably because binding to DNA is a time-dependent reaction. Fig. 2 shows that the inhibitory effect of actinomycin D on the degradation of crab poly d (A-T) was small. The inhibition was undetectable during early stages Biochim. Biophys. Acta, 157 (1968) 2o7-2o9
208
SHORT COMMUNICATIONS
2.C
1.5
~
-r 0 o z
tt ] .0
i"
1.5
~'~
-g
~
~,,/
1.0
o
z
g 0,5
=k 0 . 5
S~/t
:a
B
A o
(o
20
~'o ,'o
5~ 6o
,b
/0
3o
TIME (rain)
TIME (rain)
Fig. i. H y d r o l y s i s of t h y m u s DNA. A. , 15 AZ00m# u n i t s of D N A in 4 ml of 2. 5 mM CaCI, were i n c u b a t e d for 20 m i n a t 37 °. The p H w a s a d j u s t e d to 9.0, and the base line of N a O H u p t a k e w a s r u n for io min. The reaction w a s s t a r t e d b y the addition of o.i u n i t of micrococcal nuctease in 20/zl of o . 1 % s e r u m albumin. - - - , 15 A260m/* u n i t s of D N A in 4 ml of 2.5 mM CaCI= were i n c u b a t e d for zo min at 37 °. The p H w a s a d j u s t e d to 9.o, a n d the base line of N a O H u p t a k e w a s r u n for io min. A c t i n o m y c i n D (ioo/~g) w a s added, t h e p H r e a d j u s t e d to 9.o, and the reaction w a s s t a r t e d w i t h i n 1-2 m i n b y the a d d i t i o n of o. i u n i t of micrococcal nuclease in 2o/zl of s e r u m albumin. The procedure described in A w a s followed r o u t i n e l y in all o t h e r experiments, unless otherwise stated. B. , 15 A260m# u n i t s of DXA, 2.5 mM CaCI,, 0.2 u n i t of enzyme. Detailed procedure as in A. - - -, 15 A260m/* u n i t s of DNA, 2. 5 mM CaCI=, IOO/*g of a c t i n o m y c i n D, 0.2 u n i t of enzyme. Detailed p r o c e d u r e as in A. { } - 0 , 15 A260m# u n i t s of DNA, 2,5 mM CaC1v A c t i n o m y c i n D w a s added, the p H r e a d j u s t e d to 9.o, and t h e m i x t u r e w a s p r e i n c u b a t e d for 60 min. The reaction w a s s t a r t e d w i t h o.2 u n i t of enzyme. O t h e r details as described in A.
of hydrolysis. As the reaction proceeded, the A-T regions of the substrate were hydrolyzed preferentially and as a result the G-C regions were relatively increased. Under such conditions the inhibition by actinomycin D was detectable. During the final stages of the reaction the inhibition again disappeared. The exhaustive digests
2.0
2.0 :a:: o :~o
"TO
7°
1.0
o E :3, 0.5
o
P 0.5 :a,
o
1.0
to 2o TIME (rain)
=,o
0
1'0 20 TIME (rnin)
50
Fig. 2. H y d r o l y s i s of crab d ( A - T ) . - , 15 A260m# u n i t s of crab poly d ( A - T ) , 2.5 mM CaCI~, o,o6 u n i t of e n z y m e . Details of p r o c e d u r e as described in Fig. iA. - - - , 15 A260m# units of crab poly d ( A - T ) , 2.5 mM CaC1 v IOO/,g of a c t i n o m y c i n D, 0.o6 u n i t of enzyme. Detailed procedure as in Fig. iA. Fig. 3. H y d r o l y s i s of a m i x t u r e of c r a b p o l y d ( A - T ) (95~o) a n d t h y m u s D N A (5%). - - , I5 A260m# u n i t s of the m i x t u r e , 2.5 mM CaC12, 0.08 u n i t of enzyme. Detailed procedure as in Fig. iA. - - - , 15 A260 m/* u n i t s of the m i x t u r e , 2.5 mM CaCIz, i o o / z g of a c t i n o m y c i n D, o,08 u n i t of enzyme. Detailed procedure as in Fig. iA.
Biochim. Biophys. Acta, 157 (1968) 2o7-2o9
209
SHORT COMMUNICATIONS
contained only mononucleotides and dinucleotides in a molar ratio of 2:1. This is typical for all digests of crab poly d(A-T) by micrococcal nuclease. Fig. 3 illustrates experiments in which crab poly d(A-T) (95~) was mixed with DNA (5%). Tile inhibition by actinomycin D was greater in the presence of both substrates than when crab poly d(A-T) was the sole substrate. It seems possible that differences in susceptibility to inhibition by actinomycin D can serve as an analytical test for impurities in crab poly d(A-T) preparations. In conclusion, the "shielding" by actinomycin D of polynucleotides against degradation by micrococcal nuclease depends on the nature of the substrate and on the stage of the reaction at which the effect is measured. It seems possible that actinomycin D could be used in those cases in which the protection of G-C-containing oligonucleotide fragments from further degradation is desired. This investigation was supported by Grants E-I571 and PRP-3o from the American Cancer Society, GB-6o58 from the National Science Foundation and by Contract AT(3o-I)363o from the Atomic Energy Commission.
Laboratory o/ Enzymology, Roswell Park Memorial Institute, Bu//alo, N. Y. 142o3 (U.S.A.)
E. SULKOWSKI M. LASKOWSKI, SR.
I. H. GOLDBERG, 1VI. RABINOWITZ AND E. REICH, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2094. M. GELLERT, C. E. SMITH, D. NEVILLE AND G. FELSENFELD, J. Mol. Biol., l i (1965) 445. R. W. HYMAN AND •. DAVIDSON, Biochem. Biophys. Res. Commun., 26 (1967) 116. J. D. LEITH, Biochim. Biophys. Acta., 72 (1963) 643. B. R. McAIJSLAN, Biochem. Biophys. Res. Commun., 19 (1965) 15. L. J. ERON AND B. R. McAusLAN, Biochim. Biophys. Acta, 114 (1966) 633. N. K. SARKAR,Biochim. Biophys. Acta, I45 (1967) 174. E. SULKOWSKI AND M. LASKOWSKI, SR., J. Biol. Chem., 241 (1966) 4386. R. J. PEANASKY AND M. S z u c s , J. Biol. Chem., 239 (1964) 2525. G. M. RICHARDS, D. J. TUTAS, W. J. WECHTER AND M. LASKOWSKI, SR., Biochemistry, 6 (1967) 29o8. i i E. R. M. KAY, N. S. SIMMONS AND A. L. DOUNCE, J. Am. Chem. Soc., 74 (1952) 1724. I2 ]3. BARANOWSKI, T. BARANOWSKI, H. LASKOWSKI, Sr., European J. Biochemistry, (1968) in t h e press. I 2 3 4 5 6 7 8 9 io
Received December Ilth, 1967 Biochim. Biophys. Acta, 157 (1968) 2o7-2o 9 BBA 93297
Isolation and properties of D N A from eggs and gastrulae of Ascaris lurnbricoides In experiments on regulation processes of transcription during the early mosaic development of Ascaris, we studied the conditions for isolation of high molecular weight DNA, and some properties of the DNA preparations. The results of these experiments are described in this paper. The isolation of native DNA in substantial amounts can be achieved by a combined sodium dodecyl sulfate-chloroform-phenol procedure. The eggs or gastrulae were suspended in 0.2 M NaCl-o.I M EDTA-o.oI M Tris-HC1 solution (pH8) Biochim. Biophys. Acta, i57 (1968) 2o9-212