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Studies on the mechanism of action of micrococcal nuclease I. Degradation of thymus deoxyribonucleic acid
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BIOCHIMICA ET BIOPHYSICA ACTA
S T U D I E S ON T H E M E C H A N I S M O F A C T I O N O F MICROCOCCAL N U C L E A S E I. DEGRADATION OF THYMUS DEOXYRIBONUCLEIC ACID* W. K. ROBERTS**, C. A. D E K K E R , G. W. RUSHIZKY*** AND C. A. K N I G H T
Department o] Biochemistry and Virus Laboratory, University o] California, Berkeley, Calif. (U.S.A .) (Received October 3oth, I96I)
SUMMARY
I. A procedure involving paper electrophoresis and paper chlomatography has been adapted for use in the resolution of products resulting from the action of micrococcal nuclease on calf-thymus DNA. 2. Using this procedure, 2 7 mono-, di-, and trinucleotides have been isolated from the enzymic digest and identified. 3. With both native and heat denatured DNA, micrococcal nuclease has been found to hydrolyze preferentially those phosphodiester bonds whose cleavage results in the liberation of fragments terminating in deoxyadenylic or thymidylie acid residues bearing free 5'-hydroxyl groups. The possibility is considered that the specificity m a y be related to a lower order of structural organization resulting from sequences rich in deoxyadenylic acid and thymidylic acid residues which permits better access to the enzyme. 4. Certain trinucleotides, particularly those rich in guanylic acid, accumulate near the end of the enzymic reaction. Their further hydrolysis has been found to be slow compared with the initial hydrolysis of the entire DNA molecule. 5- The specificity of micrococcal nuclease enables it to be of some, although limited, use in the determination of base sequences in DNA.
INTRODUCTION
Micrococcal nuclease was first isolated in 1956 by CUNNINGHAM et al. 1. It has been investigated independently by several workers 2 and shown to degrade DNA3, 4, RNA 4,6 polyadenylic acid 4, polyuridylic acid s, and polycytidylic acid 5. The products in each Abbreviations: TMV, tobacco mosaic virus; A, deoxyadenosine; C, deoxycytidine; G, deoxyguanosine: T, thymidine; Ap, 3'-deoxyadenylic acid; Cp, 3'-deoxycytidylic acid; Gp, 3'deoxyguanylic acid; Tp, 3'-thymidylic acid; Kp, any of the 3'-deoxynucleotides occurring in thymus DNA. Oligonucleotides are abbreviated according to the convention of HEPPEL et al.3°; SVD, snake venom diesterase. Since this paper deals exclusively with the action of the enzyme on DNA, no a t t e m p t is made to distinguish between the corresponding ribo- and deoxyribo-derivatives. ** Present address: Department of Chemistry, King's College, Newcastle on Tyne (Great Britain ). *** Present address: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda 14, Md. (U.S.A.).
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case were mono- and dinucleotides and in some cases larger fragments. All the reaction products were found to possess a terminal 3'-phosphate, and the dinucleotides appeared to be resistant to further degradation. These studies showed that the enzyme does not have an absolute purine or pyrimidine specificity. One minor discrepancy is apparent in the results of these investigators. While CUNNINGHAM8,4 found appreciable amounts of oligonucleotides larger than dinucleotides remaining in the micrococcal nuclease digests of RNA as well as DNA, REDDI5,e found RNA to be almost completely degraded to mono- and dinucleotides. In the present study, we used a two-dimensional mapping procedure which was developed for the resolution of digests of tobacco mosaic virus RNA b y pancreatic RNAase 7, and applied it to the resolution of micrococcal nuclease digests of t h y m u s DNA. By determining the products found at various times of digestion, we have shown: (a) t h a t the enzyme has a ielative base preference for deoxyadenylic and thymidylic acid: (b) t h a t the slow degradation of trinucleotides, which accumulate near the end of the enzymic reaction, probably explains the discrepancies which have been observed in reaction products; (c) and that micrococcal nuclease is of some, although limited use in the determination of base sequences in DNA. A prelimin a r y report of p a r t of this work has been published 8. EXPERIMENTAL PROCEDURE
DNA Highly polymerized DNA was prepared from calf thymus according to the procedure of SCHWANDER AND SIGNER 9, and pleserved in citrate-sodium chloride solution at --15 °. Before use, the solution was thawed, an equal volume of ethanol added with stirring at o °, and the precipitated fibers of DNA removed with a glass rod.
Enzyme A number of pmifications of micrococcal nuclease have been described in the literaturel,~, 1°. The enzyme used in this work was a 5oo-fold purified sample prepared b y Dr. M. L. DIRKSEN in this laboratory n. I t showed no activity towards di-(pnitrophenyl) phosphate, nucleoside 2',3'-cyclic phosphates, or mononucleotides.
Enzymic digestion o[ DNA A representative digest was prepared as follows. About 60 mg of DNA were suspended in 46 ml of 0.0025 M CaC1v adjusted to p H 9 with dilute NaOH, and dissolved b y stirring overnight at 5 °. Micrococcal nuclease (25/~1 containing 537/zg protein/ml, specific activity 4 ° b y the Kunitz spectrophotometric assay) was added to 15 ml of the above stock DNA solution and the reaction was allowed to proceed at room temperature under nitrogen. Periodic additions of 0. 4 N N a O H were made at the automatic titrator to maintain the p H at 8.5~:o.5. After 8 h, the reaction temperature was raised to 4 °0 and another IO #1 of enzyme added. Four hours later the rate of alkali uptake had become very slow, and a final addition of IO/zl of enzyme had no appreciable effect. The solution was brought to p H 9.0 and allowed to incubate at 37 ° in the presence of toluene for 12 h. I t was then lyophilized, and the residue was taken up in 2 ml of water and stored at 5 ° until mapping (see below).
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This was named a Stage I I digest. A Stage I digest was prepared by adding 25 #1 of enzyme to 15 ml of the stock DNA solution and digesting analogously at room temperature. When the amount of alkali consumed was one-half of that required for the Stage I I digest, the reaction was terminated by acidifying with formic acid to p H 4, freezing, and lyophilizing. The residue was treated as described above. A Stage I I I digest was also prepared to check on the possibility of a further hydrolysis of the trinucleotides obtained in the Stage I I digest, since such a reaction might-proceed at a rate too slow for detection by the uptake of alkali. A Stage I I digest was, therefore, incubated for 48 h at 37 ° in the presence of toluene, with the adjustment of the pH to 9.0 and the addition of 15 #1 of enzyme made every 12 h. The solution was then lyophilized and treated as described above. To obtain digests (Stage liD) of denatured DNA, 15 ml of the stock DNA solution were heated for 30 min at IOO°. After cooling to 4 o°, 25/~1 of enzyme were added and the solution held at 4 °0 at a pH of 8.54-0.5. The initial rate of alkali uptake was about 20 times greater TM than in the case of native DNA digested at room temperature. Because of this, the reaction was complete (as judged by alkali uptake) after 7 h; the solution was then lyophilized and treated as above until mapping.
Mapping o/ DNA digests Aliquots of the lyophilized residues (in 2 ml of water) were mapped by applying 0.3 ml of each to Whatman 3 mm papers for electrophoresis and subsequent paper chromatography as described for RNAase digests of tobacco mosaic virus RNA v. Contact prints ~3 were then obtained from the maps.
Determination o/ the base sequences and amounts o/ compounds isolated by mapping The maps were examined under ultraviolet light, and the spots located and eluted with water. The ultraviolet light absorption 14 ratios (250/260, 280/260, 290/260 m#) at pH 2, 7, and 12, obtained from aliquots of the eluates, were then compared with those calculated for the compounds expected. The eluates were then subjected to paper chromatography with buffered ammonium sulfate v, and the absorbance (A) ratios of all isolated compounds determined. The mononucleotides weie thus identified by their absorbance ratios as well as by their RF values compared with those of standards. All absorbance determinations were made with a Beckman DU spectrophotometer. To further investigate the oligonucleotide-containing eluates, these were first freed of ammonium sulfate by adsorption on and elution from charcoal 15 as follows: about 8 ml of a neutral eluate were acidified with I N HC1 to pH 3, and sufficient acid-washed animal charcoal (Norit A) added to remove all ultraviolet-absorbing material from the solution. The charcoal was centrifuged down, washed once with water, and 8 ml of an ethanol-water-concentrated ammonia (5o:5o:1) solution were added with stirring. After 5 rain, the charcoal was sedimented and the supernatant concentrated to dryness in a warm-water bath by blowing a jet of air across it. The resulting nucleotide residue (70-9 ° °/o recovery) was then dissolved in buffer or BURTON'S reagent (see below) depending upon the method to be used for the determination of the nucleotide sequence. Where applicable, the oligonucleotides were depurinated according to the method of BURTON AND PETERSONle. To I ml of 66 % formic acid (containing 2 °/o diphenyla-
Biochim. Biophys. Acla, 55 (1962) 664-673
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mine) was added about I mg of oligonucleotide, and the solution was held for 18 h at 3 o°. The resulting purine bases and pyrimidine nucleoside mono- and diphosphates, as well as pyrimidine dinucleotides, were separated by paper chromatography. Aliquots of the solutions were applied directly to Whatman 3 mm paper, and subjected to chromatography in an isopropanol-concentrated ammonia-water (7:1:2, v/v/v) systemlL Under these conditions diphenylamine does not interfere since it moves almost to the solvent front. After reaction of a di- or trinucleotide with BURTON'S reagent or an enzyme (see below), the reaction products were chromatoglaphed in the isopropanol-ammoniawater system alongside the original di- or trinucleotide and other suitable standards. In this solvent system, the RF of an oligonucleotide depends upon its size and the number of phosphate groups presentlS,19; for example, the order of decreasing R F is cytidine, CpC, Cp, CpCp, pCp. The ultraviolet-absorbing spots were cut out, along with similar blanks, and eluted with o.oi N HC1. From the ultraviolet spectra, the identity and relative number of moles of the bases in the spots could be determined. Thus, the relative mobility of compounds, coupled with their spectrophotometric data, sufficed to identify the components of each reaction. Several oligonucleotides were hydrolyzed *° by SVD which had been purified 21 from Crotalus adamanteus venom obtained from Ross Allen's Reptile Institute, Silver Springs, Fla. As prepared, the enzyme had negligible 5'-nucleotidase activity toward adenylic acid. About I mg of oligonucleotide was dissolved in 0.5 ml of o.I M glycine buffer (pH 8.8, o.I M in magnesium acetate), 5o/,1 of the purified SVD added, and the solution held at 37 ° for 12 h. At the end of this period, an additional 25/A of SVD were added and the solution maintained at 37 ° for another 12 h. Paper chromatography using the isopropano!-ammonia-water system showed that most trinucleotides were hydrolyzed under these conditions. Dinucleotides appeared to be resistant to SVD, and were, therefore, first dephosphorylated. For this dephosphorylation, purified ** prostatic acid phosphatase (P), kindly supplied by Dr. H. FRAENKEL-CONRAT, w a s used. To about I mg of dinucleotide in 0.5 ml of o.i M sodium acetate buffer, pH 5.5, 15 #1 of enzyme were added and the solution held at 37 ° for 12 h. The dephosphorylated compound was separated from unreacted material b y paper chromatography with the same isopropanol-ammoniawater system, eluted, and treated with SVD as described above. After dephosphorylation, much less enzyme (15 #1 SVD, 37 °, 4 h) and shorter digestion periods were needed for hydrolysis.
RESULTS AND DISCUSSION
Native DNA from calf thymus was hydrolyzed with micrococcal nuclease at pH 8.5 until the uptake of alkali ceased (Stage II). Other digests were obtained by halting the enzymic hydrolysis after 50 % of the alkali required for Stage II digests had been consumed (Stage I), or by incubating Stage II digests for an additional 48 h at 37 ° with regular adjustments of pH and additions of enzyme (Stage III). Also, heat-denatured DNA was hydrolyzed by micrococcal nuclease (Stage liD) analogously to the procedure used for Stage II, i.e., until the cessation of alkali uptake. Aliquots of the various digests were then fractionated by a two-dimensional mapping procedure. Fig. I shows contact prints of the maps so obtained. B~ochim. Biophys. Acta, 55 (1962) 664-673
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The m a j o r ultraviolet-absorbing spots were then eluted from the maps and rechromatographed using a 3 ° % ammonium sulfate solvent (Table I). This procedure resolved various spots into two or more components. The total density units (A.,eom~ × volume) thus isolated amounted to 95 % of that used for mapping. All the separated compounds were characterized b y their absorption ratios at p H 2, 7, and 12; the mononucleotides were also identified by comparison of their R F values with those of standards. The base sequence of the di- and trinucleotides was determined after acid depurination or hydrolysis with snake venom diesterase
(Table II). In order to compare the amounts of the compounds present at the various
o re
Fig. I. Contact prints of various micrococcal nuclease digests of D N A fractionated b y a two-dimensional m a p p i n g procedure. For the identification of the c o m p o u n d s present in the spots see Table III. No. I, Stage I; No. 2, Stage II; No. 3, Stage IID; No. 4, Stage III.
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digestion stages, the amount of thymidylic acid found in Stage I I was raised to IOO moles, and the values of the other compounds increased in proportion. Where several compounds were present in a spot, their relative amounts (in density units) TABLE PAPER
CHROMATOGRAPHY OF
I WITH
DEOXYOLIGONUCLEOTIDES
AMMONIUM
BUFFERED
SULFATE*
IN D E S C E N T
Dinucleotide
R F value, relative to 3'-adenylic acid
CpCp TpCp TpTp TpGp CpAp CpGp ApCp GpAp ApGp TpAp GpGp ApTp ApAp
R F value, relative to 3"-adenylic acid
Trinucleotide
4. I o 1.88 1.55 1.45 1.3o i. 18 i. i o 0.96 o.87 o.78 o.70 o. 64 o.49
TpCpCp TpTpGp CpGpCp TpGpCp TpGpGp TpGpAp ApCpCp ApGpCp ApGpGp ApApGp
i. 23 1.20 1.1o i .05 1.oo o. 84 o.55 o.51 0.50 0.27
4 ° g a m m o n i u m s u l f a t e d i s s o l v e d in IOO m l of o . i M s o d i u m p h o s p h a t e , p H 7.1. TABLE IDENTIFICATION
OF O L I G O N U C L E O T I D E S
Compounds
Methods*
ApAp ApCp ApGp ApTp TpAp Cp Gp CpCp GpGp TpTp TpGp
P-SVD
TpCp ApApGp ApCpCp
P-SVD
ApGpGp ApGpCp CpGpCp TpG-pAp
BURTON
P-SVD BURTON BURTON
BURTON P-SVD P-SVD P-SVD BURTON
SVD
P-SVD P-SVD SVD SVD SVD BURTON
TpGpCp
SVD
TpTpGp
SVD
TpCpCp TpGpGp
SVD
BURTON BURTON BURTON
SVD
II
ISOLATED
FROM
CALF-THYMUS
DNA
MICROCOCCAL
NUCLEASE
DIGESTS
OF
Base ratios
pA: a d e n o s i n e = 1 : I.O 5 A d e n i n e : p C p = I : 1.o 5 Adenosine: pG = 1:1.1o A d e n i n e : p T p = I : 1.o 4 A d e n i n e : T p = i :o.96 Cp: g u a n i n e = i :1.o 9 C y t i d i n e : pC = 1 : 1.o 5 G u a n o s i n e : p G = I : 1.27 T h y m i d i n e : p T = i : 1.16 G u a n i n e : T p = I : 1.15 pC: t h y m i d i n e = I : 1.13 A d e n o s i n e : p A : p G p = i : i . o o : 1.o8 A d e n o s i n e : pC = I :2.3 A d e n o s i n e : p G = I : 2.2 A d e n o s i n e : pG : p C p = 0.8 : i . o : 1.o5 C y t i d i n e : p G + p C p = 1 : 2 . 0 8 (no pC p r e s e n t ) T h y m i d i n e a n d p G + p A p found, no p A p r e s e n t Tp: g u a n i n e : a d e n i n e = I : 1.o 3 : 1.o 7 Only t h y m i d i n e found as nucleoside G u a n i n e : Tp: p C p = i : i . o 8 : i . o 6 p T : t h y m i d i n e : p G p = 0.73 : I.O : I . I G u a n i n e : T p T p = I : 1.1 Thymidine: pC+pCp = 1:1.87 Tp: g u a n i n e = i : 1.8 Only t h y m i d i n e found as nucleoside
* " P - S V D " refers to p h o s p h a t a s e + s n a k e v e n o m dies*erase t r e a t m e n t ; "BURTON" t o a c i d i c d e p u r i n a t i o n ; " S V D " t o s n a k e v e n o m dies*erase t r e a t m e n t w i t h o u t p r i o r p h o s p h a t a s e t r e a t m e n t . F o r a d e s c r i p t i o n of t h e s e m e t h o d s , see t e x t .
Biochim.
B i o p h y s . A c t a , 55 (1962) 6 6 4 - 6 7 3
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w . K . ROBERTS, el al. TABLE III COMPOUNDS
IDENTIFIED
IN
MICROCOCCAL
NUCLEASE
DIGESTS
OF
CALF-THYMUS
DNA*
Identi/ying number on map
Relative number s* o] moles in the Stage I I digest
Stage I
Stage H D
Stage III**
Compound
Stage I I
Stage I I
Stage I I
Tp Ap Cp Gp ApTp TpAp TpTp ApAp TpGp ApCp TpCp ApGp CpCp CpGp GpGp CpAp GpAp TpGpAp ApApGp TpTpGp ApCpCp ApGpCp TpGpCp TpCpCp ApGpGp TpGpGp CpGpCp
9 I I 7 8 8 15 3 IO 2 8 6 2 6 II 2 6 It 5 14 4 5 II 6 12 13 5
ioo.o 99.1 84.0 16. 4 5.7 IO.I 13. 5 9.3 34.3 18.8 25.7 27.6 4.8 4.3 1.8 0.8 1.2 1.4 0.9 1. 7 3.9 9.o 9.6 5.6 8.1 lO.2 o. 5
o.61 0.46 o.I 9 o.13 0.46 0.44 0.40 0-37 o.29 0.26 0.25 0.23 o*** o o o o 1.2o 1.o2 0.57 o.39 o.31 o.30 o.24 0.22 o.21 o
0.88 0.93 I.OI 1.31 1.21 1.2o 1.33 1.16 1.o6 1.o4 1.1o 1.o9 1.13 1.24 1.12 o o 0.63 o 0.77 0.72 o.76 o.84 0.53 0.92 0.97 o
1.o 5 1.o3 1.32 1.64 1.35 1.o 5 1.o2 1.o 7 1.o9 I.II 1.29 I.IO 1.47 I.O 3 1.13 o 6 0.23 o o o o.I9 o. 19 o 1.o 3 o.81 o
* N a t i v e c a l f - t h y m u s D N A was digested w i t h micrococcal nuclease a t c o n s t a n t p H until the rapid u p t a k e of alkali h a d essentially s t o p p e d (Stage I I ) . F o r p u r p o s e s of c o m p a r i s o n the a m o u n t of T p released in Stage I I was a r b i t r a r i l y raised to i o o moles a n d the a m o u n t s of all o t h e r comp o u n d s increased in p r o p o r t i o n . E q u a l a m o u n t s of D N A were digested to half the alkali u p t a k e n o t e d before (Stage I), m o r e e x h a u s t i v e l y (Stage I I I ) , and following d e n a t u r a t i o n of the s u b s t r a t e (Stage I I D ) . The a m o u n t s of the c o m p o u n d s f o u n d in these digests were divided b y the a m o u n t s o b t a i n e d in the Stage I I to give the c o m p a r a t i v e ratios shown. The fraction of total b o n d s b r o k e n (as deduced f r o m alkali c o n s u m p t i o n ) was 334-2% for Stage I, 664-3 % for Stage I I and 714-3 % for Stage I I I . ** No corrections have been m a d e for h y p o c h r o m i c i t y or for nucleotides containing m e t h y l cytosine as t h e base. *** A ratio of o m e a n s t h a t a c o m p o u n d found in the Stage I I digest was n o t observed in the m a p of t h e o t h e r digest. I n these experiments, the w e a k e s t ultraviolet a b s o r b a n c e which could be observed on p a p e r c h r o m a t o g r a m s would correspond to readings on the " R e l a t i v e n u m b e r of m o l e s " scale of a p p r o x . 1. 5 for a mononucleotide, o.75 for a dinucleotide, a n d 0. 5 for a trinucleotide.
were obtained from the ratio of their A 26om~ values after separation b y paper chrom a t o g r a p h y with the ammonium sulfate system. The molar amounts were then calculated using the appropriate extinction coefficients. For comparison, the relative number of moles of each compound found on the maps of the various digests was divided b y the corresponding value obtained from the Stage I I digest (Table III). In Table III, the mono-, di- and trinucleotides are listed in order of decreasing amounts released after a Stage I digestion. Examination of the Stage I/Stage I I column shows that micrococcal nuclease first liberates from thymus DNA those compounds which are richest in adenylic and thymidylic acid. Thus, the dinucleotides B i o c h i m . B i o p h y s . A c t a , 55 (1962) 664-673
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composed of Ap and/or Tp residues show a greater percentage liberated at Stage I than those with only one Ap or Tp residue, and this is greater than for those with none. A similar relationship holds for the trinucleotides. Further evidence of an ApTp preference can be found in the structures and relative number of moles of the diand trinucleotides at Stage II, where about 95 % of these compounds possess Ap or Tp at the 5'-hydroxyl end. Several possible modes of action have been considered for micrococcal nuclease which might explain the above results: (a) The enzyme has a preference for the bonds -XpA- and -XpT- regardless of whether the A and T moieties terminate in a 3'-monophosphate or he within a polynucleotide sequence. (b) Similar to (a) with the additional postulate that the newly formed oligonucleotides terminating in -Xp are rapidly digested when X = A or T, slowly when X = C and very slowly when X = G, the attack being primarily but not exclusively exonucleolytie. (c) Similar to (b) but with the further quahfication that initial attack occurs in regions of the molecule which have a high density of Ap and Tp residues. Proposal (a) fails to account for the very rapid and preferential hberation of the mononucleotides, Tp and Ap, during the early stages of digestion. Proposal (b) does not adequately explain the preferential early release of those dinucleotides composed of Ap and/or Tp and of those trinucleotides possessing more than one Ap or Tp residue. Proposal (c) best accommodates the data presented here and in the accompanying paper; additional support for it has been derived from a study of the effect of secondary structure on the kinetics 1~. One can define a nuclease as an endo-esterase if it attacks the middle of a nucleic acid chain to liberate oligonucleotides, and as an exo-esterase if it attacks the end of a chain to hberate mononucleotides. Micrococcal nuclease thus first acts as an endoesterase to hydrolyse a DNA molecule in regions of high Ap and Tp concentrations. The large oligonucleotides produced are further degraded by a combination of endoand exo-esterase attack, both showing a preference for -XpA and -XpT- bonds. This leads to an initial liberation of mono-, di-, and trinucleotides which are rich in Ap and Tp. Eventually, the less favored bonds are hydrolyzed releasing the Cp and Gp nucleotides until at the end of Stage II only those trinucleotides which are particularly resistant to exo-esterase attack (terminating in Cp or Gp) are left as potential substrates. Upon prolonged digestion (Stage III) most of these are also hydrolyzed, as can be seen from the Stage III/Stage II column in Table III. Since micrococcal nuclease hydrolyzes heat-denatured DNA at a rate up to IOO times faster than for native DNA lz, and since the ease of denaturation of a DNA molecule increases with increasing AT/CG ratio, it was considered possible that the enzyme might attack at regions of high Ap and Tp density because of a localized lower order of structural organization. If this were true, prior denaturation of the substrate might be expected to markedly alter the distribution of products obtained on hydrolysis, increasing the Cp and Gp at the expense of some of the Ap and Tp. An examination of the Stage l i D / S t a g e I I column (Table rlI) shows that the opening up of the DNA molecule makes phosphodiester bonds more available, i.e., Stage l i D falls between Stages II and I I I in extent of hydrolysis. However, denaturation hardly changes the preference of the enzyme (Table III, also compare Fig. I, No. 2 and 3). This could mean that the physical state of the substrate does not affect the distribution of the products obtained, or that the physical state of the substrate after heating Biochim. Biophys. Acta, 55 (1962) 664-673
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w, K. ROBERTS, et al.
and cooling is not sufficiently altered to affect the nature of the bonds attacked. I t should be recalled that much of the original hypochromicity of a DNA sample is regained after cooling, particularly in the presence of divalent cations such as Ca 2+, and that this is normally interpreted in terms of a restored, while random, base-base interaction. The results of the action of micrococcal nuclease on heat-denatured DNA should thus be compared with the results on TMV-RNA presented in the accompanying paper ~. I t is interesting to compare the Ap-Tp preference of micrococcal nuclease with the specificity observed b y VAtqECKO AND LASKOWSKI~ for splenic DNAase II. The latter enzyme also liberates nucleotides terminating in a 3'-phosphate, but preferentially splits bonds of the type -TpX- and -ApX-. Thus, micrococcal nuclease hydrolyzes DNA to give oligonucleotides with Tp or Ap at the 5'-hydroxyl end, while splenic DNAase yields oligonucleotides most of which have Tp or Ap at the 3'-phosphate end. Our results concerning the preference of micrococcal nuclease extend the independent but more limited observations of POCHON AND PRIVAT DE GARILHE on the products arising from the action of this enzyme on DNA, which were interpreted in terms of a preference for only -XpT- bonds 25. One of the reasons for engaging in this work was to determine whether micrococcal nuclease would be of value for the elucidation of nucleotide sequences in DNA. Although the existence and frequency of occurrence of certain nucleotide sequences in sevelal DNA preparations has been established b y enzymic 2s and chemicaW, *s methods, progress has been hampered b y the lack of nucleases with strict specificity. While micrococcal nuclease shows a low substrate specificity, new oligonucleotides were obtained with the enzyme. The accumulation of products of digestion larger than dinucleotides proved to be a problem of dynamics; for example, trinucleotides were formed from larger compounds and at the same time further degraded to monoand dinucleotides. This dynamic aspect m a y explain the previously mentioned discrepancies in reaction products obtained from micrococcal nuclease digests 4-e, which would thus be due to differences in completion of digestion rather than differences in enzyme. The preference of the enzyme greatly reduced the possible number of diand trinucleotides found in substantial amount (i.e., more than 5 relative moles, Table III) in the DNA digest. This specificity, which is more pronounced in the case of DNA than RNA 2~, leads to the accumulation of TpGpGp, for example, and not other trinucleotides, such as TpApAp, which are more susceptible to enzymic hydrolysis. As a result, dinucleotides and relatively resistant trinucleotides listed in Table I I I should be found in the Stage I I digest of any DNA containing these sequences. The amount found would not be quantitative, of course, but would only represent a minimum value for the sequence. However, in the case of the trinucleotides ApGpGp and TpGpGp, which are hydrolyzed very slowly, if at all, the value obtained is probably a good estimation of the occurrence of this sequence in the DNA molecule. I t is apparent from Table I I I that in the hydrolysis of DNA by micrococcal nuclease the sequence X p G is particularly intractable. For example, the electrophoresis band near the origin of Fig. I, No. I, is quite rich in Gp and it is probable that much of the 5 % loss in density units during the mapping of Stage I I digests is due to undetected Gp-rich oligonucleotides. However, this specificity allows an estimate of the occurrence of the various X p G sequences in calf-thymus DNA. Adding together the Biochim. Biophys. Acta, 55 (1962) 664-673
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r e l a t i v e n u m b e r o f m o l e s o f X p G s e q u e n c e s (for e a c h o f t h e 4 b a s e s ) a n d d i v i d i n g b y t h e t o t a l n u m b e r of m o l e s of G p , g i v e s t h e f o l l o w i n g r e s u l t s : T p G ---- 3 9 ~ , A p G = 3 1 % , G p G ---- 14 ~/o, C p G = 3 % , a n d X p G ---- 13 % ( h e r e X is u n k n o w n , a s i n t h e case of Gp, GpGp, and GpAp). This calculation, although hampered by the fact t h a t X p G is u n k n o w n a n d t h a t s o m e G p o l i g o n u c l e o t i d e s w e r e p r o b a b l y l o s t o n t h e m a p , c a n b e c o m p a r e d w i t h t h e r e s u l t s of JOSSE, KAISER AND KORNBERG zg. T h e s e workers found that DNA, synthesized enzymically using native calf-thymus DNA as primer, had the following relative XpG ratios: TpG = 36 %, ApG = 34 ~, GpG = 23 % , a n d C p G = 7 ~ . I t c a n b e s e e n t h a t t h e s e r e s u l t s a g r e e i n a g e n e r a l w a y , a n d thus provide some direct evidence that biosynthesized DNA contains the same sequences as "natural" DNA. ACKNOWLEDGEMENTS This work was suppo, ted in part by a grant from the National Science Foundation, a n d a r e s e a r c h g r a n t , E - 6 3 4 , f r o m T h e N a t i o n a l I n s t i t u t e of A l l e r g y a n d I n f e c t i o u s D i s e a s e s , N a t i o n a l I n s t i t u t e s of H e a l t h , U . S . P u b l i c H e a l t h S e r v i c e . W e t h a n k D r . J . D. SMITH w h o h a s k i n d l y m a d e a v a i l a b l e t h e r e s u l t s of h i s u n p u b l i s h e d s t u d i e s c o n d u c t e d i n 1957 . REFERENCES
J. Am. Chem. Soc., 78 (i956) 4642. j . D. SMITH, personal communication. L. CUNNXNGHAM, J. Am. Chem. Soc., 80 (r958) 2546. L. CUr~mNGHAM, Ann. N. Y. Acad. Sci., 8i (I959) 788. K. K. REDDI, Biochim. Biophys. Acta, 36 (I959) r32. K. K. REDDI, Proc. Natl. Acad. Sci. U.S., 45 (I959) 293. G. W. RUSHIZKY AND C. A. KNIGHT, Virology, I I (I960) 236. G. W. RUSHIZKY, C. A. KNIGHT, W. K. ROBERTS AND C. A. DEKKER, Biochem. Biophys. Research Communs., 2 (I96o) I53. 8 H. SCHWANDER AND R. SIGNER, Helv. Chim. Acta, 33 (I95o) I52L 10 M. P~IVAT DE GARILHE, G. FASSINA, F. POCHON AND J. PILLEr, Bull. soc. chem. biol., 4 ° (1958) r9o5. 11 M. L. DIRKSEN, P h . D . Thesis, University of California, Berkeley, I959. a2 M. L. DIRKSEN AND C. A. DEKKER, Biochem. Biophys. Research Communs., 2 (196o) r47. is K. C. SMITH AND F. W. ALLEN, J. A m . Chem. Soc., 75 (I953) 213I. 14 G. H. BEAVEN, E. R. HOLIDAY AND E. A. JOHNSON, in E. CHAR~AFF ArrD J. N. DAVIDSON, The Nucleic Acids, Vol. I, Academic Press, Inc., New York, I955, P. 493. is R. K. CRANE AND F. LrpMANN, J. Biol. Chem., 2oi (1953) 253. is K. BUETON AND G. B. PETEESF.N, Biochim. Biophys. Acta, 26 (I957) 667. 1~ R. MARKHAM AND J. D. SMrTH, Biochem. J., 52 (I952) 558, 565 . 18 W. E. RAZZELL AND H. G. KHORANA, J. Biol. Chem., 234 (I959) 2 r i 4 . 19 W. E. RAZZELL AND H. G. KHOEANA, J. Biol. Chem., 236 (I96I) II44. 20 F. FELIX, J. L. POTTER AND M. LASKOWSKI, J. Biol. Chem., 235 (I96O) II5O. sx j . F. KOERNER AND R. L. SINSHEIMER, J. Biol. Chem., 228 (I957) IO49. s2 W. OSTROWSKI AND A. TSUGITA, Arch. Biochem. Biophys., 94 (I96I) 68. ~s Cr. W. RUSHIZKY, C. A. KNIGHT, W. K. ROB~-RTS AND C. A. DEKKER, Biochim. Biophys. Acta, 55 (r962) 674. s4 S. VANECICOAND M. LASKOWSKL J. Biol. Chem., 236 (I96I) II35. ~5 F. POCHON AND M. PRIVAT DE GARXLHE, Bull. soc. chim. biol., 42 (x96o) 795ss R. L. SINSHEIMER, J. Biol. Chem., 2i 5 (I955) 579s, H. S. SHAPIEO AND E. CHARGAFE, Biochim. Biophys. Acta, 26 (I957) 608; 39 (I96O) 68. s8 K. BURTON AND G. B. PETERSEN, Biochem. J., 75 (I96O) 17. s9 j . JossE, A. D. KAXSEE AND A. KOENBERG, J. Biol. Chem., 236 (I96I) 864. so L. A. HEPPEL, P. R. WHITFmLD AND R. MARKHAM, Biochem. J., 60 (r955) 8. I L. CUNNINGHAM, B . W . CATLIN AND M . PRIVAT DE GARILHE,
2 s 4 5 e 7 s
Biochim. Biophys. A a a , 55 (i962) 664-673
BIOCHIMICA ET BIOPHYSICA ACTA
S T U D I E S ON T H E M E C H A N I S M O F A C T I O N O F MICROCOCCAL N U C L E A S E I. DEGRADATION OF THYMUS DEOXYRIBONUCLEIC ACID* W. K. ROBERTS**, C. A. D E K K E R , G. W. RUSHIZKY*** AND C. A. K N I G H T
Department o] Biochemistry and Virus Laboratory, University o] California, Berkeley, Calif. (U.S.A .) (Received October 3oth, I96I)
SUMMARY
I. A procedure involving paper electrophoresis and paper chlomatography has been adapted for use in the resolution of products resulting from the action of micrococcal nuclease on calf-thymus DNA. 2. Using this procedure, 2 7 mono-, di-, and trinucleotides have been isolated from the enzymic digest and identified. 3. With both native and heat denatured DNA, micrococcal nuclease has been found to hydrolyze preferentially those phosphodiester bonds whose cleavage results in the liberation of fragments terminating in deoxyadenylic or thymidylie acid residues bearing free 5'-hydroxyl groups. The possibility is considered that the specificity m a y be related to a lower order of structural organization resulting from sequences rich in deoxyadenylic acid and thymidylic acid residues which permits better access to the enzyme. 4. Certain trinucleotides, particularly those rich in guanylic acid, accumulate near the end of the enzymic reaction. Their further hydrolysis has been found to be slow compared with the initial hydrolysis of the entire DNA molecule. 5- The specificity of micrococcal nuclease enables it to be of some, although limited, use in the determination of base sequences in DNA.
INTRODUCTION
Micrococcal nuclease was first isolated in 1956 by CUNNINGHAM et al. 1. It has been investigated independently by several workers 2 and shown to degrade DNA3, 4, RNA 4,6 polyadenylic acid 4, polyuridylic acid s, and polycytidylic acid 5. The products in each Abbreviations: TMV, tobacco mosaic virus; A, deoxyadenosine; C, deoxycytidine; G, deoxyguanosine: T, thymidine; Ap, 3'-deoxyadenylic acid; Cp, 3'-deoxycytidylic acid; Gp, 3'deoxyguanylic acid; Tp, 3'-thymidylic acid; Kp, any of the 3'-deoxynucleotides occurring in thymus DNA. Oligonucleotides are abbreviated according to the convention of HEPPEL et al.3°; SVD, snake venom diesterase. Since this paper deals exclusively with the action of the enzyme on DNA, no a t t e m p t is made to distinguish between the corresponding ribo- and deoxyribo-derivatives. ** Present address: Department of Chemistry, King's College, Newcastle on Tyne (Great Britain ). *** Present address: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda 14, Md. (U.S.A.).
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case were mono- and dinucleotides and in some cases larger fragments. All the reaction products were found to possess a terminal 3'-phosphate, and the dinucleotides appeared to be resistant to further degradation. These studies showed that the enzyme does not have an absolute purine or pyrimidine specificity. One minor discrepancy is apparent in the results of these investigators. While CUNNINGHAM8,4 found appreciable amounts of oligonucleotides larger than dinucleotides remaining in the micrococcal nuclease digests of RNA as well as DNA, REDDI5,e found RNA to be almost completely degraded to mono- and dinucleotides. In the present study, we used a two-dimensional mapping procedure which was developed for the resolution of digests of tobacco mosaic virus RNA b y pancreatic RNAase 7, and applied it to the resolution of micrococcal nuclease digests of t h y m u s DNA. By determining the products found at various times of digestion, we have shown: (a) t h a t the enzyme has a ielative base preference for deoxyadenylic and thymidylic acid: (b) t h a t the slow degradation of trinucleotides, which accumulate near the end of the enzymic reaction, probably explains the discrepancies which have been observed in reaction products; (c) and that micrococcal nuclease is of some, although limited use in the determination of base sequences in DNA. A prelimin a r y report of p a r t of this work has been published 8. EXPERIMENTAL PROCEDURE
DNA Highly polymerized DNA was prepared from calf thymus according to the procedure of SCHWANDER AND SIGNER 9, and pleserved in citrate-sodium chloride solution at --15 °. Before use, the solution was thawed, an equal volume of ethanol added with stirring at o °, and the precipitated fibers of DNA removed with a glass rod.
Enzyme A number of pmifications of micrococcal nuclease have been described in the literaturel,~, 1°. The enzyme used in this work was a 5oo-fold purified sample prepared b y Dr. M. L. DIRKSEN in this laboratory n. I t showed no activity towards di-(pnitrophenyl) phosphate, nucleoside 2',3'-cyclic phosphates, or mononucleotides.
Enzymic digestion o[ DNA A representative digest was prepared as follows. About 60 mg of DNA were suspended in 46 ml of 0.0025 M CaC1v adjusted to p H 9 with dilute NaOH, and dissolved b y stirring overnight at 5 °. Micrococcal nuclease (25/~1 containing 537/zg protein/ml, specific activity 4 ° b y the Kunitz spectrophotometric assay) was added to 15 ml of the above stock DNA solution and the reaction was allowed to proceed at room temperature under nitrogen. Periodic additions of 0. 4 N N a O H were made at the automatic titrator to maintain the p H at 8.5~:o.5. After 8 h, the reaction temperature was raised to 4 °0 and another IO #1 of enzyme added. Four hours later the rate of alkali uptake had become very slow, and a final addition of IO/zl of enzyme had no appreciable effect. The solution was brought to p H 9.0 and allowed to incubate at 37 ° in the presence of toluene for 12 h. I t was then lyophilized, and the residue was taken up in 2 ml of water and stored at 5 ° until mapping (see below).
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This was named a Stage I I digest. A Stage I digest was prepared by adding 25 #1 of enzyme to 15 ml of the stock DNA solution and digesting analogously at room temperature. When the amount of alkali consumed was one-half of that required for the Stage I I digest, the reaction was terminated by acidifying with formic acid to p H 4, freezing, and lyophilizing. The residue was treated as described above. A Stage I I I digest was also prepared to check on the possibility of a further hydrolysis of the trinucleotides obtained in the Stage I I digest, since such a reaction might-proceed at a rate too slow for detection by the uptake of alkali. A Stage I I digest was, therefore, incubated for 48 h at 37 ° in the presence of toluene, with the adjustment of the pH to 9.0 and the addition of 15 #1 of enzyme made every 12 h. The solution was then lyophilized and treated as described above. To obtain digests (Stage liD) of denatured DNA, 15 ml of the stock DNA solution were heated for 30 min at IOO°. After cooling to 4 o°, 25/~1 of enzyme were added and the solution held at 4 °0 at a pH of 8.54-0.5. The initial rate of alkali uptake was about 20 times greater TM than in the case of native DNA digested at room temperature. Because of this, the reaction was complete (as judged by alkali uptake) after 7 h; the solution was then lyophilized and treated as above until mapping.
Mapping o/ DNA digests Aliquots of the lyophilized residues (in 2 ml of water) were mapped by applying 0.3 ml of each to Whatman 3 mm papers for electrophoresis and subsequent paper chromatography as described for RNAase digests of tobacco mosaic virus RNA v. Contact prints ~3 were then obtained from the maps.
Determination o/ the base sequences and amounts o/ compounds isolated by mapping The maps were examined under ultraviolet light, and the spots located and eluted with water. The ultraviolet light absorption 14 ratios (250/260, 280/260, 290/260 m#) at pH 2, 7, and 12, obtained from aliquots of the eluates, were then compared with those calculated for the compounds expected. The eluates were then subjected to paper chromatography with buffered ammonium sulfate v, and the absorbance (A) ratios of all isolated compounds determined. The mononucleotides weie thus identified by their absorbance ratios as well as by their RF values compared with those of standards. All absorbance determinations were made with a Beckman DU spectrophotometer. To further investigate the oligonucleotide-containing eluates, these were first freed of ammonium sulfate by adsorption on and elution from charcoal 15 as follows: about 8 ml of a neutral eluate were acidified with I N HC1 to pH 3, and sufficient acid-washed animal charcoal (Norit A) added to remove all ultraviolet-absorbing material from the solution. The charcoal was centrifuged down, washed once with water, and 8 ml of an ethanol-water-concentrated ammonia (5o:5o:1) solution were added with stirring. After 5 rain, the charcoal was sedimented and the supernatant concentrated to dryness in a warm-water bath by blowing a jet of air across it. The resulting nucleotide residue (70-9 ° °/o recovery) was then dissolved in buffer or BURTON'S reagent (see below) depending upon the method to be used for the determination of the nucleotide sequence. Where applicable, the oligonucleotides were depurinated according to the method of BURTON AND PETERSONle. To I ml of 66 % formic acid (containing 2 °/o diphenyla-
Biochim. Biophys. Acla, 55 (1962) 664-673
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mine) was added about I mg of oligonucleotide, and the solution was held for 18 h at 3 o°. The resulting purine bases and pyrimidine nucleoside mono- and diphosphates, as well as pyrimidine dinucleotides, were separated by paper chromatography. Aliquots of the solutions were applied directly to Whatman 3 mm paper, and subjected to chromatography in an isopropanol-concentrated ammonia-water (7:1:2, v/v/v) systemlL Under these conditions diphenylamine does not interfere since it moves almost to the solvent front. After reaction of a di- or trinucleotide with BURTON'S reagent or an enzyme (see below), the reaction products were chromatoglaphed in the isopropanol-ammoniawater system alongside the original di- or trinucleotide and other suitable standards. In this solvent system, the RF of an oligonucleotide depends upon its size and the number of phosphate groups presentlS,19; for example, the order of decreasing R F is cytidine, CpC, Cp, CpCp, pCp. The ultraviolet-absorbing spots were cut out, along with similar blanks, and eluted with o.oi N HC1. From the ultraviolet spectra, the identity and relative number of moles of the bases in the spots could be determined. Thus, the relative mobility of compounds, coupled with their spectrophotometric data, sufficed to identify the components of each reaction. Several oligonucleotides were hydrolyzed *° by SVD which had been purified 21 from Crotalus adamanteus venom obtained from Ross Allen's Reptile Institute, Silver Springs, Fla. As prepared, the enzyme had negligible 5'-nucleotidase activity toward adenylic acid. About I mg of oligonucleotide was dissolved in 0.5 ml of o.I M glycine buffer (pH 8.8, o.I M in magnesium acetate), 5o/,1 of the purified SVD added, and the solution held at 37 ° for 12 h. At the end of this period, an additional 25/A of SVD were added and the solution maintained at 37 ° for another 12 h. Paper chromatography using the isopropano!-ammonia-water system showed that most trinucleotides were hydrolyzed under these conditions. Dinucleotides appeared to be resistant to SVD, and were, therefore, first dephosphorylated. For this dephosphorylation, purified ** prostatic acid phosphatase (P), kindly supplied by Dr. H. FRAENKEL-CONRAT, w a s used. To about I mg of dinucleotide in 0.5 ml of o.i M sodium acetate buffer, pH 5.5, 15 #1 of enzyme were added and the solution held at 37 ° for 12 h. The dephosphorylated compound was separated from unreacted material b y paper chromatography with the same isopropanol-ammoniawater system, eluted, and treated with SVD as described above. After dephosphorylation, much less enzyme (15 #1 SVD, 37 °, 4 h) and shorter digestion periods were needed for hydrolysis.
RESULTS AND DISCUSSION
Native DNA from calf thymus was hydrolyzed with micrococcal nuclease at pH 8.5 until the uptake of alkali ceased (Stage II). Other digests were obtained by halting the enzymic hydrolysis after 50 % of the alkali required for Stage II digests had been consumed (Stage I), or by incubating Stage II digests for an additional 48 h at 37 ° with regular adjustments of pH and additions of enzyme (Stage III). Also, heat-denatured DNA was hydrolyzed by micrococcal nuclease (Stage liD) analogously to the procedure used for Stage II, i.e., until the cessation of alkali uptake. Aliquots of the various digests were then fractionated by a two-dimensional mapping procedure. Fig. I shows contact prints of the maps so obtained. B~ochim. Biophys. Acta, 55 (1962) 664-673
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w . K . ROBERTS, et al.
The m a j o r ultraviolet-absorbing spots were then eluted from the maps and rechromatographed using a 3 ° % ammonium sulfate solvent (Table I). This procedure resolved various spots into two or more components. The total density units (A.,eom~ × volume) thus isolated amounted to 95 % of that used for mapping. All the separated compounds were characterized b y their absorption ratios at p H 2, 7, and 12; the mononucleotides were also identified by comparison of their R F values with those of standards. The base sequence of the di- and trinucleotides was determined after acid depurination or hydrolysis with snake venom diesterase
(Table II). In order to compare the amounts of the compounds present at the various
o re
Fig. I. Contact prints of various micrococcal nuclease digests of D N A fractionated b y a two-dimensional m a p p i n g procedure. For the identification of the c o m p o u n d s present in the spots see Table III. No. I, Stage I; No. 2, Stage II; No. 3, Stage IID; No. 4, Stage III.
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digestion stages, the amount of thymidylic acid found in Stage I I was raised to IOO moles, and the values of the other compounds increased in proportion. Where several compounds were present in a spot, their relative amounts (in density units) TABLE PAPER
CHROMATOGRAPHY OF
I WITH
DEOXYOLIGONUCLEOTIDES
AMMONIUM
BUFFERED
SULFATE*
IN D E S C E N T
Dinucleotide
R F value, relative to 3'-adenylic acid
CpCp TpCp TpTp TpGp CpAp CpGp ApCp GpAp ApGp TpAp GpGp ApTp ApAp
R F value, relative to 3"-adenylic acid
Trinucleotide
4. I o 1.88 1.55 1.45 1.3o i. 18 i. i o 0.96 o.87 o.78 o.70 o. 64 o.49
TpCpCp TpTpGp CpGpCp TpGpCp TpGpGp TpGpAp ApCpCp ApGpCp ApGpGp ApApGp
i. 23 1.20 1.1o i .05 1.oo o. 84 o.55 o.51 0.50 0.27
4 ° g a m m o n i u m s u l f a t e d i s s o l v e d in IOO m l of o . i M s o d i u m p h o s p h a t e , p H 7.1. TABLE IDENTIFICATION
OF O L I G O N U C L E O T I D E S
Compounds
Methods*
ApAp ApCp ApGp ApTp TpAp Cp Gp CpCp GpGp TpTp TpGp
P-SVD
TpCp ApApGp ApCpCp
P-SVD
ApGpGp ApGpCp CpGpCp TpG-pAp
BURTON
P-SVD BURTON BURTON
BURTON P-SVD P-SVD P-SVD BURTON
SVD
P-SVD P-SVD SVD SVD SVD BURTON
TpGpCp
SVD
TpTpGp
SVD
TpCpCp TpGpGp
SVD
BURTON BURTON BURTON
SVD
II
ISOLATED
FROM
CALF-THYMUS
DNA
MICROCOCCAL
NUCLEASE
DIGESTS
OF
Base ratios
pA: a d e n o s i n e = 1 : I.O 5 A d e n i n e : p C p = I : 1.o 5 Adenosine: pG = 1:1.1o A d e n i n e : p T p = I : 1.o 4 A d e n i n e : T p = i :o.96 Cp: g u a n i n e = i :1.o 9 C y t i d i n e : pC = 1 : 1.o 5 G u a n o s i n e : p G = I : 1.27 T h y m i d i n e : p T = i : 1.16 G u a n i n e : T p = I : 1.15 pC: t h y m i d i n e = I : 1.13 A d e n o s i n e : p A : p G p = i : i . o o : 1.o8 A d e n o s i n e : pC = I :2.3 A d e n o s i n e : p G = I : 2.2 A d e n o s i n e : pG : p C p = 0.8 : i . o : 1.o5 C y t i d i n e : p G + p C p = 1 : 2 . 0 8 (no pC p r e s e n t ) T h y m i d i n e a n d p G + p A p found, no p A p r e s e n t Tp: g u a n i n e : a d e n i n e = I : 1.o 3 : 1.o 7 Only t h y m i d i n e found as nucleoside G u a n i n e : Tp: p C p = i : i . o 8 : i . o 6 p T : t h y m i d i n e : p G p = 0.73 : I.O : I . I G u a n i n e : T p T p = I : 1.1 Thymidine: pC+pCp = 1:1.87 Tp: g u a n i n e = i : 1.8 Only t h y m i d i n e found as nucleoside
* " P - S V D " refers to p h o s p h a t a s e + s n a k e v e n o m dies*erase t r e a t m e n t ; "BURTON" t o a c i d i c d e p u r i n a t i o n ; " S V D " t o s n a k e v e n o m dies*erase t r e a t m e n t w i t h o u t p r i o r p h o s p h a t a s e t r e a t m e n t . F o r a d e s c r i p t i o n of t h e s e m e t h o d s , see t e x t .
Biochim.
B i o p h y s . A c t a , 55 (1962) 6 6 4 - 6 7 3
670
w . K . ROBERTS, el al. TABLE III COMPOUNDS
IDENTIFIED
IN
MICROCOCCAL
NUCLEASE
DIGESTS
OF
CALF-THYMUS
DNA*
Identi/ying number on map
Relative number s* o] moles in the Stage I I digest
Stage I
Stage H D
Stage III**
Compound
Stage I I
Stage I I
Stage I I
Tp Ap Cp Gp ApTp TpAp TpTp ApAp TpGp ApCp TpCp ApGp CpCp CpGp GpGp CpAp GpAp TpGpAp ApApGp TpTpGp ApCpCp ApGpCp TpGpCp TpCpCp ApGpGp TpGpGp CpGpCp
9 I I 7 8 8 15 3 IO 2 8 6 2 6 II 2 6 It 5 14 4 5 II 6 12 13 5
ioo.o 99.1 84.0 16. 4 5.7 IO.I 13. 5 9.3 34.3 18.8 25.7 27.6 4.8 4.3 1.8 0.8 1.2 1.4 0.9 1. 7 3.9 9.o 9.6 5.6 8.1 lO.2 o. 5
o.61 0.46 o.I 9 o.13 0.46 0.44 0.40 0-37 o.29 0.26 0.25 0.23 o*** o o o o 1.2o 1.o2 0.57 o.39 o.31 o.30 o.24 0.22 o.21 o
0.88 0.93 I.OI 1.31 1.21 1.2o 1.33 1.16 1.o6 1.o4 1.1o 1.o9 1.13 1.24 1.12 o o 0.63 o 0.77 0.72 o.76 o.84 0.53 0.92 0.97 o
1.o 5 1.o3 1.32 1.64 1.35 1.o 5 1.o2 1.o 7 1.o9 I.II 1.29 I.IO 1.47 I.O 3 1.13 o 6 0.23 o o o o.I9 o. 19 o 1.o 3 o.81 o
* N a t i v e c a l f - t h y m u s D N A was digested w i t h micrococcal nuclease a t c o n s t a n t p H until the rapid u p t a k e of alkali h a d essentially s t o p p e d (Stage I I ) . F o r p u r p o s e s of c o m p a r i s o n the a m o u n t of T p released in Stage I I was a r b i t r a r i l y raised to i o o moles a n d the a m o u n t s of all o t h e r comp o u n d s increased in p r o p o r t i o n . E q u a l a m o u n t s of D N A were digested to half the alkali u p t a k e n o t e d before (Stage I), m o r e e x h a u s t i v e l y (Stage I I I ) , and following d e n a t u r a t i o n of the s u b s t r a t e (Stage I I D ) . The a m o u n t s of the c o m p o u n d s f o u n d in these digests were divided b y the a m o u n t s o b t a i n e d in the Stage I I to give the c o m p a r a t i v e ratios shown. The fraction of total b o n d s b r o k e n (as deduced f r o m alkali c o n s u m p t i o n ) was 334-2% for Stage I, 664-3 % for Stage I I and 714-3 % for Stage I I I . ** No corrections have been m a d e for h y p o c h r o m i c i t y or for nucleotides containing m e t h y l cytosine as t h e base. *** A ratio of o m e a n s t h a t a c o m p o u n d found in the Stage I I digest was n o t observed in the m a p of t h e o t h e r digest. I n these experiments, the w e a k e s t ultraviolet a b s o r b a n c e which could be observed on p a p e r c h r o m a t o g r a m s would correspond to readings on the " R e l a t i v e n u m b e r of m o l e s " scale of a p p r o x . 1. 5 for a mononucleotide, o.75 for a dinucleotide, a n d 0. 5 for a trinucleotide.
were obtained from the ratio of their A 26om~ values after separation b y paper chrom a t o g r a p h y with the ammonium sulfate system. The molar amounts were then calculated using the appropriate extinction coefficients. For comparison, the relative number of moles of each compound found on the maps of the various digests was divided b y the corresponding value obtained from the Stage I I digest (Table III). In Table III, the mono-, di- and trinucleotides are listed in order of decreasing amounts released after a Stage I digestion. Examination of the Stage I/Stage I I column shows that micrococcal nuclease first liberates from thymus DNA those compounds which are richest in adenylic and thymidylic acid. Thus, the dinucleotides B i o c h i m . B i o p h y s . A c t a , 55 (1962) 664-673
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composed of Ap and/or Tp residues show a greater percentage liberated at Stage I than those with only one Ap or Tp residue, and this is greater than for those with none. A similar relationship holds for the trinucleotides. Further evidence of an ApTp preference can be found in the structures and relative number of moles of the diand trinucleotides at Stage II, where about 95 % of these compounds possess Ap or Tp at the 5'-hydroxyl end. Several possible modes of action have been considered for micrococcal nuclease which might explain the above results: (a) The enzyme has a preference for the bonds -XpA- and -XpT- regardless of whether the A and T moieties terminate in a 3'-monophosphate or he within a polynucleotide sequence. (b) Similar to (a) with the additional postulate that the newly formed oligonucleotides terminating in -Xp are rapidly digested when X = A or T, slowly when X = C and very slowly when X = G, the attack being primarily but not exclusively exonucleolytie. (c) Similar to (b) but with the further quahfication that initial attack occurs in regions of the molecule which have a high density of Ap and Tp residues. Proposal (a) fails to account for the very rapid and preferential hberation of the mononucleotides, Tp and Ap, during the early stages of digestion. Proposal (b) does not adequately explain the preferential early release of those dinucleotides composed of Ap and/or Tp and of those trinucleotides possessing more than one Ap or Tp residue. Proposal (c) best accommodates the data presented here and in the accompanying paper; additional support for it has been derived from a study of the effect of secondary structure on the kinetics 1~. One can define a nuclease as an endo-esterase if it attacks the middle of a nucleic acid chain to liberate oligonucleotides, and as an exo-esterase if it attacks the end of a chain to hberate mononucleotides. Micrococcal nuclease thus first acts as an endoesterase to hydrolyse a DNA molecule in regions of high Ap and Tp concentrations. The large oligonucleotides produced are further degraded by a combination of endoand exo-esterase attack, both showing a preference for -XpA and -XpT- bonds. This leads to an initial liberation of mono-, di-, and trinucleotides which are rich in Ap and Tp. Eventually, the less favored bonds are hydrolyzed releasing the Cp and Gp nucleotides until at the end of Stage II only those trinucleotides which are particularly resistant to exo-esterase attack (terminating in Cp or Gp) are left as potential substrates. Upon prolonged digestion (Stage III) most of these are also hydrolyzed, as can be seen from the Stage III/Stage II column in Table III. Since micrococcal nuclease hydrolyzes heat-denatured DNA at a rate up to IOO times faster than for native DNA lz, and since the ease of denaturation of a DNA molecule increases with increasing AT/CG ratio, it was considered possible that the enzyme might attack at regions of high Ap and Tp density because of a localized lower order of structural organization. If this were true, prior denaturation of the substrate might be expected to markedly alter the distribution of products obtained on hydrolysis, increasing the Cp and Gp at the expense of some of the Ap and Tp. An examination of the Stage l i D / S t a g e I I column (Table rlI) shows that the opening up of the DNA molecule makes phosphodiester bonds more available, i.e., Stage l i D falls between Stages II and I I I in extent of hydrolysis. However, denaturation hardly changes the preference of the enzyme (Table III, also compare Fig. I, No. 2 and 3). This could mean that the physical state of the substrate does not affect the distribution of the products obtained, or that the physical state of the substrate after heating Biochim. Biophys. Acta, 55 (1962) 664-673
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w, K. ROBERTS, et al.
and cooling is not sufficiently altered to affect the nature of the bonds attacked. I t should be recalled that much of the original hypochromicity of a DNA sample is regained after cooling, particularly in the presence of divalent cations such as Ca 2+, and that this is normally interpreted in terms of a restored, while random, base-base interaction. The results of the action of micrococcal nuclease on heat-denatured DNA should thus be compared with the results on TMV-RNA presented in the accompanying paper ~. I t is interesting to compare the Ap-Tp preference of micrococcal nuclease with the specificity observed b y VAtqECKO AND LASKOWSKI~ for splenic DNAase II. The latter enzyme also liberates nucleotides terminating in a 3'-phosphate, but preferentially splits bonds of the type -TpX- and -ApX-. Thus, micrococcal nuclease hydrolyzes DNA to give oligonucleotides with Tp or Ap at the 5'-hydroxyl end, while splenic DNAase yields oligonucleotides most of which have Tp or Ap at the 3'-phosphate end. Our results concerning the preference of micrococcal nuclease extend the independent but more limited observations of POCHON AND PRIVAT DE GARILHE on the products arising from the action of this enzyme on DNA, which were interpreted in terms of a preference for only -XpT- bonds 25. One of the reasons for engaging in this work was to determine whether micrococcal nuclease would be of value for the elucidation of nucleotide sequences in DNA. Although the existence and frequency of occurrence of certain nucleotide sequences in sevelal DNA preparations has been established b y enzymic 2s and chemicaW, *s methods, progress has been hampered b y the lack of nucleases with strict specificity. While micrococcal nuclease shows a low substrate specificity, new oligonucleotides were obtained with the enzyme. The accumulation of products of digestion larger than dinucleotides proved to be a problem of dynamics; for example, trinucleotides were formed from larger compounds and at the same time further degraded to monoand dinucleotides. This dynamic aspect m a y explain the previously mentioned discrepancies in reaction products obtained from micrococcal nuclease digests 4-e, which would thus be due to differences in completion of digestion rather than differences in enzyme. The preference of the enzyme greatly reduced the possible number of diand trinucleotides found in substantial amount (i.e., more than 5 relative moles, Table III) in the DNA digest. This specificity, which is more pronounced in the case of DNA than RNA 2~, leads to the accumulation of TpGpGp, for example, and not other trinucleotides, such as TpApAp, which are more susceptible to enzymic hydrolysis. As a result, dinucleotides and relatively resistant trinucleotides listed in Table I I I should be found in the Stage I I digest of any DNA containing these sequences. The amount found would not be quantitative, of course, but would only represent a minimum value for the sequence. However, in the case of the trinucleotides ApGpGp and TpGpGp, which are hydrolyzed very slowly, if at all, the value obtained is probably a good estimation of the occurrence of this sequence in the DNA molecule. I t is apparent from Table I I I that in the hydrolysis of DNA by micrococcal nuclease the sequence X p G is particularly intractable. For example, the electrophoresis band near the origin of Fig. I, No. I, is quite rich in Gp and it is probable that much of the 5 % loss in density units during the mapping of Stage I I digests is due to undetected Gp-rich oligonucleotides. However, this specificity allows an estimate of the occurrence of the various X p G sequences in calf-thymus DNA. Adding together the Biochim. Biophys. Acta, 55 (1962) 664-673
MECHANISM OF ACTION OF MICROCOCCAL NUCLEASE.
I
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r e l a t i v e n u m b e r o f m o l e s o f X p G s e q u e n c e s (for e a c h o f t h e 4 b a s e s ) a n d d i v i d i n g b y t h e t o t a l n u m b e r of m o l e s of G p , g i v e s t h e f o l l o w i n g r e s u l t s : T p G ---- 3 9 ~ , A p G = 3 1 % , G p G ---- 14 ~/o, C p G = 3 % , a n d X p G ---- 13 % ( h e r e X is u n k n o w n , a s i n t h e case of Gp, GpGp, and GpAp). This calculation, although hampered by the fact t h a t X p G is u n k n o w n a n d t h a t s o m e G p o l i g o n u c l e o t i d e s w e r e p r o b a b l y l o s t o n t h e m a p , c a n b e c o m p a r e d w i t h t h e r e s u l t s of JOSSE, KAISER AND KORNBERG zg. T h e s e workers found that DNA, synthesized enzymically using native calf-thymus DNA as primer, had the following relative XpG ratios: TpG = 36 %, ApG = 34 ~, GpG = 23 % , a n d C p G = 7 ~ . I t c a n b e s e e n t h a t t h e s e r e s u l t s a g r e e i n a g e n e r a l w a y , a n d thus provide some direct evidence that biosynthesized DNA contains the same sequences as "natural" DNA. ACKNOWLEDGEMENTS This work was suppo, ted in part by a grant from the National Science Foundation, a n d a r e s e a r c h g r a n t , E - 6 3 4 , f r o m T h e N a t i o n a l I n s t i t u t e of A l l e r g y a n d I n f e c t i o u s D i s e a s e s , N a t i o n a l I n s t i t u t e s of H e a l t h , U . S . P u b l i c H e a l t h S e r v i c e . W e t h a n k D r . J . D. SMITH w h o h a s k i n d l y m a d e a v a i l a b l e t h e r e s u l t s of h i s u n p u b l i s h e d s t u d i e s c o n d u c t e d i n 1957 . REFERENCES
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