- Email: [email protected]
Breakdown of DNA stimulated by nucleoside di- and triphosphates in cell-free extracts of Mycobacterium smegmatis
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
215
NOTES
BBA 911~A. B r e a k d o w n of D N A s t i m u l a t e d by nuc|eoside di- and triphosphates in cell-free extracts of Mycobacterium smegrnatis
It has been observed that incubation of cell-free extracts of Mycobacterium smegmatis results in a substantial formation of soluble deoxyribonucleotide, the deoxyribose of which originates from the acid-insoluble material in the extracts 1. The investigations reported in this communication indicate that this is due to a breakdown of DNA stimulated by ribonucleoside di- or triphosphates, rather than to deoxyribosyl transfer as previously suggested. The growth medium was prepared as described by RATLEDGE AND W I N D E R 2, except that magnesium citrate was replaced by 0.5 g of MgSO4. 7 H20/1 and that carbon was made limiting by reducing the glycerol content to 2.5 ml/1. It contained 2 mg of Fe2+/l. Cultures were grown at 37 ° on a rotary shaker and cells were usually harvested after 35 h (start of decelerating phase) and stored at about --IO °. Extracts were prepared by suspending the cells in 0.05 M Tris buffer, pH 8.0, containing I mM mercaptoethanol, treating for 2 min near o ° with a Ioo-W ultrasonic generator, centrifuging at 25 ooo ×g for 2o rain near o °, and discarding the sediment. Samples (0.8 ml) of the extract were incubated at 37 ° with 20/,moles of MgC12 and, where appropriate, 4 #moles of ribonucleotide in a total vol. of i ml. The reaction was stopped by adding 2 ml of N HCIO4, the mixture was centrifuged and the acidsoluble supernatant was removed from the acid-insoluble residue. The acid-soluble fraction was adjusted to pH 9.0 and was incubated with 0.5 mg of Crotalus adamanteus venom in order to dephosphorylate the deoxyribonucleotides a. The acid-insoluble fraction was incubated with I ml of 0.3 M KOH at 37 ° for I h, the pH was adiusted to 7.0; 50/,moles of MgC12 and 0.5 mg of pancreatic deoxyribonuclease were added, it was incubated at 37 ° for 2 h, adjusted to pH 9.0 and incubated with 0.5 mg of Crotalus adamanteus venom as a source of phosphodiesterase and monoesterase for 1. 5 h. The deoxyribonucleosides liberated in both fractions were assayed microbiologically4. Deoxyribose was estimated by a slight modification of the diphenylamine method of GILES AND MYERS5 after bromination and alkaline hydrolysis 6. Protein was measured by the biuret method. The results in Table I show that incubation of cell-free extracts of M. smegmatis in buffer in the presence of ATP led to the formation of a substantial amount of soluble deoxyribonucleotide. Formation of soluble deoxyribonucleotide took place also when extracts were incubated without ATP, pairtcularly when the extracts were from recently harvested cells, but this activity declined more rapidly on storage of the cells than did the activity with ATP. The time course of the reactions suggested that the ATP was broken down during them and that the activity in the absence of added ATP was due to ATP already in the extracts. ADP, CDP and CTP had much the same effect as ATP in stimulating the formation of soluble deoxyribonucleotide, while adenine, cytosine, adenosine, cytidine, AMP, CMP, deoxylibose, orthophosphate and pyrophosphate were without effect.
Biochim. Biophys. Acta, 134 (1967) 215-217
216 TABLE
PRELIMINARY
NOTES
I
FORMATION
OF ACID-SOLUBLE
DEOXYRIBONUCLEOTIDE
DURING
INCUBATION
OF EXTRACTS
OF M.
smegmatis Cells were stored a t - I O ° for v a r i o u s periods before p r e p a r a t i o n of the extracts.
Addition to
Period o/
Soluble deoxyribonucleotide
incubation mixture
incubation
(ml~moles/mg protein)
(rain)
............................. Period o/storage o/cells • week
2 weeks
4 weeks
None
o 3°
9.8 41.o
6.8 25.4
9.9 15.3
ATP
o 3°
11.4 95.0
6.3 7t.9
9-9 62.1
The products formed were nucleotide rather than nucleoside, since they were adsorbed on anion exchange resin, but it is not yet known whether they were nucleoside mono-, di- or triphosphates, or short-chain oligonucleotides. Most of the effect, at least, was not due to a ribonucleotide reductase system: much the same ratio of purine-bound to pyrimidine-bound deoxyribose was observed in the soluble fraction when ATP was used as when CTP was used; no requirement for a reducing system could be found, and all activity could be removed by streptomycin treatment. This conclusion was confirmed by the finding that, when deoxyribose appeared in the acid-soluble fraction after incubation with ATP, almost exactly the same amount disappeared from the acid-insoluble fraction (Table II).
TABLE
II
EFFECT
OF
INCUBATION
DEOXYRIBOSE
ON
IN EXTRACTS
ACID-SOLUBLE OF
M.
AND
INSOLUBLE
DEOXYRIBONUCLEOTIDE
AND
TOTAL
smegmatis
Cells w e r e 4 ° h old. T h e y w e r e stored for 4 w e e k s at - l O % T h e
s o l u b l e fraction was treated w i t h snake v e n o m , and t h e insoluble fraction w i t h d e o x y r i b o n u c l e a s e and snake v e n o m , b e f o r e m i c r o b i o l o g i c a l a s s a y o f d e o x y r i b o n u c l e o s i d e and m e a s u r e m e n t of d e o x y r i b o s e by b r o m i n a t i o n , alkaline h y d r o l y s i s and r e a c t i o n w i t h d i p h e n y l a m i n e .
Addition to
Period o/
Deoxyribonucleotide
Total deoxyribose
incubation mixture
incubation
(mizmoles/mg protein)
(re#moles/rag protein)
Acid-soluble traction
Acid-insolu-
Acid-soluble
Acid-insolu-
ble [raction
/raction
ble /raction
None
ATP
(rain)
o 30
Change
9.9 I5.3 i 5.4
397 374 .... 23
9.4 20.6 ~ 11.2
426 407 19
Change
9.9 62.1 + 52.2
405 325 -- 80
lO.4 83 .8 + 73.4
437 358 -- 79
o 3°
When the acid-insoluble fraction was incubated with deoxyribonuclease and snake venom in order to convert the D N A to deoxyribonucleosides which were then estimated microbiologically, it was found that the changes in amount of DNA so Bioehim. Biophys. Acta, 134 (1967) 2 I 5 2~ 7
2I 7
PRELIMINARY NOTES
determined were equal to the changes in amount of deoxyribose in the insoluble fraction (Table II). This showed that the soluble deoxyribonucleotide was not formed b y deoxyribosyl transfer from a form of acid-insoluble deoxyribose other than DNA. Hence the formation of soluble deoxyribonucleotide in the presence of ribonucleoside di- and triphosphates was due to a breakdown of DNA stimulated by these substances. Thus, this organism contains a very active enzyme capable of breaking down DNA in the presence of nucleoside di- or triphosphates. This enzyme is very similar to one reported from 3/[icrococcus lysodeikticus ~ and hence presumably occurs in a number of microorganisms at least. It is not known what significance it has in the metabolism of such organisms, but its presence is obviously an important factor in studies on DNA and deoxyribonucleotide metabolism in cell-free extracts derived from them. This work was assisted by a grant from the Medical Research Council of Ireland, and M. P. COUGHLANreceived a maintenance allowance from the Department of Education, Republic of Ireland.
Department o~ Biochemistry, Trinity College, Dublin (Ireland)
F R A N K G. W I N D E R MICHAEL P . COUGHLAN
F. G. WINDER AND M. P. COUGHLAN, Biochem. J., IOO (1966) 39P. C. RATLEDGE AND F. G. WINDER, Biochem. J., 84 (1962) 5Ol. A. LARSSON, Jr. Biol. Chem., 238 (1963) 3414 . E. HOFF-JORGENSEN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, p. 781. 5 K. GILES AND A. MYERS, Nature, 2o6 (1965) 93. 6 1~. L. BLAKLEY, J. Biol. Chem., 241 (1966) 176. 7 Y- TSUDA AND B. S. STRAUSS, Biochemistry, 3 (1964) 1678. I 2 3 4
Received August I5th, 1966 Biochim. Biophys. Acta, i 3 4 (1967) 215-217
BBA 91145
SilicageI-Dfinnschichtchromatographie von Nucleosiden, Nucleotiden und Oligonucleotiden Die Dfinnschichtchromatographie der obengenannten Substanzen an Celluloseschichten ffihrt sehr oft zu ausgezeichneten Trennungen und bedeutet ein wichtiges analytisches HilfsmitteP -3. Leider war es nicht m6glich, dieses Trennverfahred in den pr~parativen MalDstab zu fibertragen. Bei der schrittweisen, chemischen Synthese yon Oligonucleotiden werden h/~ufig in geeigneter Weise geschtitzte Nucleotide und Oligonucleotide in gr613erer Menge ben6tigt. Die gew6hnlich zur Reinigung benutzte DEAE-Cellulose-Anionenaustauscherchromatographie ffihrt nicht immer zu reineD Verbindungen und ist aulDerdem zeitraubend. Ftir die Darstellung und Biochim. Biophys. Acta, 134 (1967) 217-22o
PRELIMINARY
215
NOTES
BBA 911~A. B r e a k d o w n of D N A s t i m u l a t e d by nuc|eoside di- and triphosphates in cell-free extracts of Mycobacterium smegrnatis
It has been observed that incubation of cell-free extracts of Mycobacterium smegmatis results in a substantial formation of soluble deoxyribonucleotide, the deoxyribose of which originates from the acid-insoluble material in the extracts 1. The investigations reported in this communication indicate that this is due to a breakdown of DNA stimulated by ribonucleoside di- or triphosphates, rather than to deoxyribosyl transfer as previously suggested. The growth medium was prepared as described by RATLEDGE AND W I N D E R 2, except that magnesium citrate was replaced by 0.5 g of MgSO4. 7 H20/1 and that carbon was made limiting by reducing the glycerol content to 2.5 ml/1. It contained 2 mg of Fe2+/l. Cultures were grown at 37 ° on a rotary shaker and cells were usually harvested after 35 h (start of decelerating phase) and stored at about --IO °. Extracts were prepared by suspending the cells in 0.05 M Tris buffer, pH 8.0, containing I mM mercaptoethanol, treating for 2 min near o ° with a Ioo-W ultrasonic generator, centrifuging at 25 ooo ×g for 2o rain near o °, and discarding the sediment. Samples (0.8 ml) of the extract were incubated at 37 ° with 20/,moles of MgC12 and, where appropriate, 4 #moles of ribonucleotide in a total vol. of i ml. The reaction was stopped by adding 2 ml of N HCIO4, the mixture was centrifuged and the acidsoluble supernatant was removed from the acid-insoluble residue. The acid-soluble fraction was adjusted to pH 9.0 and was incubated with 0.5 mg of Crotalus adamanteus venom in order to dephosphorylate the deoxyribonucleotides a. The acid-insoluble fraction was incubated with I ml of 0.3 M KOH at 37 ° for I h, the pH was adiusted to 7.0; 50/,moles of MgC12 and 0.5 mg of pancreatic deoxyribonuclease were added, it was incubated at 37 ° for 2 h, adjusted to pH 9.0 and incubated with 0.5 mg of Crotalus adamanteus venom as a source of phosphodiesterase and monoesterase for 1. 5 h. The deoxyribonucleosides liberated in both fractions were assayed microbiologically4. Deoxyribose was estimated by a slight modification of the diphenylamine method of GILES AND MYERS5 after bromination and alkaline hydrolysis 6. Protein was measured by the biuret method. The results in Table I show that incubation of cell-free extracts of M. smegmatis in buffer in the presence of ATP led to the formation of a substantial amount of soluble deoxyribonucleotide. Formation of soluble deoxyribonucleotide took place also when extracts were incubated without ATP, pairtcularly when the extracts were from recently harvested cells, but this activity declined more rapidly on storage of the cells than did the activity with ATP. The time course of the reactions suggested that the ATP was broken down during them and that the activity in the absence of added ATP was due to ATP already in the extracts. ADP, CDP and CTP had much the same effect as ATP in stimulating the formation of soluble deoxyribonucleotide, while adenine, cytosine, adenosine, cytidine, AMP, CMP, deoxylibose, orthophosphate and pyrophosphate were without effect.
Biochim. Biophys. Acta, 134 (1967) 215-217
216 TABLE
PRELIMINARY
NOTES
I
FORMATION
OF ACID-SOLUBLE
DEOXYRIBONUCLEOTIDE
DURING
INCUBATION
OF EXTRACTS
OF M.
smegmatis Cells were stored a t - I O ° for v a r i o u s periods before p r e p a r a t i o n of the extracts.
Addition to
Period o/
Soluble deoxyribonucleotide
incubation mixture
incubation
(ml~moles/mg protein)
(rain)
............................. Period o/storage o/cells • week
2 weeks
4 weeks
None
o 3°
9.8 41.o
6.8 25.4
9.9 15.3
ATP
o 3°
11.4 95.0
6.3 7t.9
9-9 62.1
The products formed were nucleotide rather than nucleoside, since they were adsorbed on anion exchange resin, but it is not yet known whether they were nucleoside mono-, di- or triphosphates, or short-chain oligonucleotides. Most of the effect, at least, was not due to a ribonucleotide reductase system: much the same ratio of purine-bound to pyrimidine-bound deoxyribose was observed in the soluble fraction when ATP was used as when CTP was used; no requirement for a reducing system could be found, and all activity could be removed by streptomycin treatment. This conclusion was confirmed by the finding that, when deoxyribose appeared in the acid-soluble fraction after incubation with ATP, almost exactly the same amount disappeared from the acid-insoluble fraction (Table II).
TABLE
II
EFFECT
OF
INCUBATION
DEOXYRIBOSE
ON
IN EXTRACTS
ACID-SOLUBLE OF
M.
AND
INSOLUBLE
DEOXYRIBONUCLEOTIDE
AND
TOTAL
smegmatis
Cells w e r e 4 ° h old. T h e y w e r e stored for 4 w e e k s at - l O % T h e
s o l u b l e fraction was treated w i t h snake v e n o m , and t h e insoluble fraction w i t h d e o x y r i b o n u c l e a s e and snake v e n o m , b e f o r e m i c r o b i o l o g i c a l a s s a y o f d e o x y r i b o n u c l e o s i d e and m e a s u r e m e n t of d e o x y r i b o s e by b r o m i n a t i o n , alkaline h y d r o l y s i s and r e a c t i o n w i t h d i p h e n y l a m i n e .
Addition to
Period o/
Deoxyribonucleotide
Total deoxyribose
incubation mixture
incubation
(mizmoles/mg protein)
(re#moles/rag protein)
Acid-soluble traction
Acid-insolu-
Acid-soluble
Acid-insolu-
ble [raction
/raction
ble /raction
None
ATP
(rain)
o 30
Change
9.9 I5.3 i 5.4
397 374 .... 23
9.4 20.6 ~ 11.2
426 407 19
Change
9.9 62.1 + 52.2
405 325 -- 80
lO.4 83 .8 + 73.4
437 358 -- 79
o 3°
When the acid-insoluble fraction was incubated with deoxyribonuclease and snake venom in order to convert the D N A to deoxyribonucleosides which were then estimated microbiologically, it was found that the changes in amount of DNA so Bioehim. Biophys. Acta, 134 (1967) 2 I 5 2~ 7
2I 7
PRELIMINARY NOTES
determined were equal to the changes in amount of deoxyribose in the insoluble fraction (Table II). This showed that the soluble deoxyribonucleotide was not formed b y deoxyribosyl transfer from a form of acid-insoluble deoxyribose other than DNA. Hence the formation of soluble deoxyribonucleotide in the presence of ribonucleoside di- and triphosphates was due to a breakdown of DNA stimulated by these substances. Thus, this organism contains a very active enzyme capable of breaking down DNA in the presence of nucleoside di- or triphosphates. This enzyme is very similar to one reported from 3/[icrococcus lysodeikticus ~ and hence presumably occurs in a number of microorganisms at least. It is not known what significance it has in the metabolism of such organisms, but its presence is obviously an important factor in studies on DNA and deoxyribonucleotide metabolism in cell-free extracts derived from them. This work was assisted by a grant from the Medical Research Council of Ireland, and M. P. COUGHLANreceived a maintenance allowance from the Department of Education, Republic of Ireland.
Department o~ Biochemistry, Trinity College, Dublin (Ireland)
F R A N K G. W I N D E R MICHAEL P . COUGHLAN
F. G. WINDER AND M. P. COUGHLAN, Biochem. J., IOO (1966) 39P. C. RATLEDGE AND F. G. WINDER, Biochem. J., 84 (1962) 5Ol. A. LARSSON, Jr. Biol. Chem., 238 (1963) 3414 . E. HOFF-JORGENSEN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, N e w York, 1957, p. 781. 5 K. GILES AND A. MYERS, Nature, 2o6 (1965) 93. 6 1~. L. BLAKLEY, J. Biol. Chem., 241 (1966) 176. 7 Y- TSUDA AND B. S. STRAUSS, Biochemistry, 3 (1964) 1678. I 2 3 4
Received August I5th, 1966 Biochim. Biophys. Acta, i 3 4 (1967) 215-217
BBA 91145
SilicageI-Dfinnschichtchromatographie von Nucleosiden, Nucleotiden und Oligonucleotiden Die Dfinnschichtchromatographie der obengenannten Substanzen an Celluloseschichten ffihrt sehr oft zu ausgezeichneten Trennungen und bedeutet ein wichtiges analytisches HilfsmitteP -3. Leider war es nicht m6glich, dieses Trennverfahred in den pr~parativen MalDstab zu fibertragen. Bei der schrittweisen, chemischen Synthese yon Oligonucleotiden werden h/~ufig in geeigneter Weise geschtitzte Nucleotide und Oligonucleotide in gr613erer Menge ben6tigt. Die gew6hnlich zur Reinigung benutzte DEAE-Cellulose-Anionenaustauscherchromatographie ffihrt nicht immer zu reineD Verbindungen und ist aulDerdem zeitraubend. Ftir die Darstellung und Biochim. Biophys. Acta, 134 (1967) 217-22o