Properties of an ATP-dependent deoxyribonuclease from Micrococcus luteus. A characterisation of the reaction products

166

Biochimica et Biophysica Acta, 340 (1974) 166--176 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 97934 P R O P E R T I E S OF AN A T P- D E PE N D E N T D E O X Y R I B O N U C L E A S E FROM M I C R O C O C C U S L U T E U S . A C H A R A C T E R I S A T I O N OF T H E REACTION PRODUCTS

BOB VAN DORP*, MARIAN Th.E. CAULEN and PETER H. POUWELS Medical Biological Laboratory TNO, Rijswijk 2100 (The Netherlands) (Received October 23rd, 1973) Summary In the early stage of degradation of T7 DNA by the ATP-dependent DNAase f r o m M i c r o c o c c u s luteus the following products are formed: 1. Acid-soluble, dialysable material representing approxi m at el y 35% of the degradation products. 2. Material of relatively low molecular weight and of heterogeneous size ( f r o m a few to a b o u t 300 nucleotides long) which is mainly acid-insoluble and represents 15--20% of the degradation products. These molecules are for a large part single-stranded, but a fraction, or possibly all of them, contains a doublestranded region which may be as long as 150--300 nucleotides. 3. Material of relatively high molecular weight consisting of molecules with single-stranded regions (on the average 80 nucleotides long) at their ends.

Introduction It is generally believed t h a t the A T P - d e p e n d e n t DNAases f o u n d in a number of microorganisms are involved in genetic recombination. The precise functions of the enzymes are however, n o t known. T he e n z y m e from M i c r o c o c c u s luteus, which is the object of our study, degrades double-stranded linear DNA exonucleolytically [1, 2] . Single-stranded linear DNA is degraded at a greatly reduced rate and circular double-stranded DNA is completely resistant to attack by the e n z y m e [ 1, 3] . T he e n z y m e degrades a DNA molecule completely before a n o t h e r molecule is attacked and bot h strands are degraded at the same time. When an excess of e n z y m e is used and incubations are p e r f o r m e d at low t e m p e r a t u r e [3] or with a limiting a m o u n t of ATP [ 4 ] , partially degraded DNA molecules can be isolated. U pon centrifugation through sucrose gradients, the released p r o d u ct s are f o u n d near the t op of the gradient and the degradation of the DNA molecules becomes evident from a shift of their position towards the t o p of the gradient. * Laboratory

of Moleculax

Genetics, Leiden State University, Leiden, T h e Netherlands.

167 After prolonged incubation of the DNA with the enzyme, all material is converted into acid-soluble oligonucleotides [2]. These observations prompted us to investigate the nature of the reaction products which are formed at an early stage of the reaction. In the present paper we describe the isolation and characterization of the slowly sedimenting degradation products and the partly degraded DNA molecules. Materials and Methods

De termination of A TP-dependent DNAase activity The enzyme preparations used were DNA--agarose fractions, obtained as previously described [3]. These preparations are approximately 50% pure as judged by gel electrophoresis and contain no endo- or exonuclease activity on double-stranded DNA in the absence of ATP. The activity on single-stranded DNA is only 4% of the nuclease activity found on double-stranded DNA. Preparation of T7[ 3 2 P ] D N A has been described [3]. The DNA obtained in this way contains less than 5 single-strand breaks per molecule. The ATP-dependent DNAase activity was assayed by measuring the 32p made acid-soluble by incubation of T7[ 32p] DNA with the enzyme. The reaction mixture (0.4 ml) contained: 20 pmoles Tris--HC1 (pH 9.0}, 10 pmoles MgSO4, 0.4 pmole ATP, 2 pg DNA and 10-6--10 -s unit of enzyme (see below). The reaction was started by addition of 0.1 ml of the enzyme solution. Incubation was continued for 30 min at 30 ° C. One unit of the enzyme is defined as the amount of enzyme which renders 10 pmoles of DNA phosphorus acidsoluble in 1 min under the assay conditions. Isolation of small products and intermediates T 7 1 3 2 p ] D N A with a specific activity of a b o u t 3.104 cpm per pg was degraded with an excess of enzyme (1.7.10 -4 unit/pg DNA) at 0°C for 8 min, under conditions as described in the previous paragraph, or for 15 min at 30°C with a limiting concentration (42 pM) of ATP [4]. Under these conditions approximately 50% of the DNA is degraded. The reaction was stopped by addition of phenol and shaking. The upper, aqueous phase was dialysed against 0.01 M Tris--HC1 (pH 7.5), 0.05 M NaC1 and centrifuged through sucrose gradients for 15 h at 2 4 0 0 0 rev./min in a spinco SW 27 rotor in order to separate the partly degraded DNA molecules (intermediates) from the products of low molecular weight (Fig. 1). The intermediates and the nonsedimenting products were pooled separately. From the original material approximately 85% was recovered from the gradients. Intermediates and nonsedimenting products were dialysed against 0.01 M Tris--HC1 (pH 7.5}, 0.05 M NaC1, concentrated by freeze-drying and dialyzed once more against the same buffer. During dialysis approximately 70% of the products of low molecular weight were lost as oligonucleotides containing less than 5 nucleotides [5]. The resulting preparation of nonsedimenting products will hereafter be called small products. Enzyme incubations The assay for terminal phosphate with alkaline phosphatase was performed as described by Furlong [6]. Exonuclease I from Escherichia coli was

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purified on DEAE-cellulose and DNA-agarose columns. Incubations with the e n z y m e were performed as described by Lehman and Nussbaum [7]. Aspergillus nuclease $1 was purified from a-amylase powder up to the DEAE step according to Vogt [8]. Incubations with the enzyme were carried out as described by the same author, T4 DNA polymerase was purified from T4-infected E. coli bacteria by chromatography on DNA-agarose and DEAE-cellulose columns. Incubations with the enzyme were performed as described by Goulian, Lucas and Kornberg [9] with approximately 1 #g of DNA and 1 unit of enzyme in the incubation mixture. The specific activity of the [3 H] dTTP used was 5 • 102 Ci/M. The m e t h o d of incubation with exonuclease III has been described previously [3]. 32p was counted on planchets in a Nuclear Chicago planchet counter. Filters with 3 H-labelled DNA were counted, after immersion in toluene with scintillator, in a Nuclear Chicago scintillation counter. Results

Detection o f single-stranded material in the reaction products Nitrocellulose filters retain single-stranded but not double-stranded DNA [10]. To investigate whether the products, which are formed during limited degradation of T7[ 32 P]DNA by the ATP-dependent DNAase ofM. luteus, are at least partially single-stranded, we filtered the material through nitrocellulose membranes. We found that, depending on the incubation conditions used, 30--80% of the radioactive material was bound to the filter, even when the DNA had been deproteinized with phenol or a combination of sodium dodecylsulphate and pronase. Less than 7.5% of native T7 DNA was bound to the

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FRACTION NUMBER Fig. 2. Binding b y n i t r o c e l l u l o s e filters of t h e v a r i o u s r e a c t i o n p r o d u c t s as s e p a r a t e d b y sucrose g r a d i e n t c e n t r i f u g a t i o n . A f t e r partial d e g r a d a t i o n of T 7 1 3 2 p ] D N A w i t h an e x c e s s o f A T P - d e p e n d e n t D N A a s e (5 rain at 0 ° C ) t h e r e a c t i o n p r o d u c t s w e r e d e p r o t e i n i z e d w i t h p h e n o l , d i a l y s e d and c e n t r i f u g e d t h r o u g h a sucrose g r a d i e n t f o r 15 h at 2 5 0 0 0 r e v . / m i n . E v e r y three f r a c t i o n s w e r e c o m b i n e d , d i l u t e d in 5 m l 1 M NaC1, 0 . 0 1 M Tris--HC1 ( p H 8.0) and filtered t h r o u g h n i t r o c e l l u l o s e filters ( S a r t o r i u s SM 1 1 3 0 7 ) . T h e filters w e r e r i n s e d w i t h 5 m l o f t h e s a m e b u f f e r . T h e h i s t o g r a m s h o w s the p e r c e n t a g e o f the t o t a l r a d i o a c t i v i t y w h i c h was r e t a i n e d b y t h e filter. T h e c u r v e r e p r e s e n t s t h e t o t a l r a d i o a c t i v i t y .

filter. In order to investigate which type of reaction product binds to nitrocellulose we first separated the products by sucrose gradient centrifugation into small products and intermediates {Fig. 2) and then determined the retention by nitrocellulose for the various fractions. The results in Fig. 2 show that small products and intermediates are bound to nitrocellulose approximately to the same extent, with the exception of the material in the top fractions. This material contains probably also very small oligonucleotides, which are not retained by the filter, even when single-stranded. These results suggest that both the intermediates and the small products are single-stranded or contain singlestranded regions. In a control experiment we observed that T7 DNA which had been degraded with exonuclease III from E. coli for 4.7%, and thus contained only 4.7% single-stranded material, was quantitatively bound to nitrocellulose. To determine whether the intermediates of high molecular weight which were retained for a large part by the nitrocellulose filters, were partially or fully single-stranded, we centrifuged them to equilibrium in CsC1. Double- and singlestranded T7 DNA were used as density markers. We found only one band of radioactive material at the position of the double-stranded marker DNA, indicating that the single-stranded regions in the intermediates must be relatively small. The "small products" of the reaction did not band in CsC1, probably because they are too short.

Characterization of the small products We have previously reported [3] that the reaction products formed at 30 and at 0°C are different. To further analyse this difference experiments were carried o u t with small products formed at either 0 or 30°C. After isolation of the products by means of sucrose gradient centrifugation and dialysis to remove mono- and oligonucleotides with a chain length smaller than 5 nucleotides [5] (see Materials and Methods), we determined the average chain length with alkaline phosphatase. The average chain lenght of the products of a degradation at 30°C was 10.8 nucleotides whereas the products formed at 0°C were

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F i g . 3. S e p h a d e x G - 2 0 0 a n a l y s i s o f s m a l l p r o d u c t s p r o d u c e d a t 0 ° C . 1 m l o f a s o l u t i o n o f s m a l l p r o d u c t s to which tRNA and ATP were added as m a r k e r s , was applied to a Sephadex G-200 column (45 cm X 1 cm). The material was eluted with 0.01 M Tris--HCl (pH 7.5), 0.05 M NaCl. For every fraction the total radioactivity ( ) and the percentage acid-soluble material (...... ) were determined. The position of the ATP and tRNA markers was determined by measuring the absorption at 260 nm. For some fractions the S value was also determined (see arrows). (a) Untreated small products. (b) S m a l l p r o d u c t s d e n a t u r e d b y h e a t i n g a t 1 0 0 ° C f o r 5 r a i n a n d c o o l e d o n i c e . (c) S m a l l p r o d u c t s i n c u b a t e d w i t h 1 u n i t Aspergillus n u c l e a s e S 1 ( e x c e s s ) f o r 1 5 r a i n a t 4 5 ° C .

on the average 3.5 nucleotides longer. T he size distribution of the product s f o r m e d was investigated by gel filtration on Sephadex G-200 with ATP and t R N A as markers (Fig. 3a). A large q u a n t i t y of the material was f o u n d at the exclusion volume. T he sedimentation coefficient of this material, det erm i ned by centrifugation in a sucrose gradient with transfer RNA as a marker (4.0 S)

171 was found to be 5.8 S. The point of 50% acid solubility gives the position of molecules with a chain length of 17 nucleotides [5]. When the small products were heated to 100°C before gel filtration to denature any double-stranded material present, we observed a shift of the material towards lower molecular weight (Fig. 3b). This indicates that at least part of the material is doublestranded. After denaturation very large fragments were still present with a sedimentation coefficient up to 5.6 S. This value corresponds to a molecular weight of 9 . 1 0 4 for single-stranded DNA [11]. To investigate which fraction of the small products is double-stranded and to determine the size distribution of the double-stranded fragments, small products were digested with the single-strand-specific Aspergillus nuclease S1 [8] and then passed through a G-200 column (Fig. 3c). Incubation with this enzyme caused a significant change of the elution profile. More than 50% of the radioactivity was found at the position of the ATP marker, but very little at the position corresponding to oligonucleotides {fractions between t R N A and ATP). Of the material which was eluted before t R N A a substantial fraction was resistant to the enzyme and thus represents doublestranded DNA. Part of this material is of high molecular weight since it is eluted at the exclusion volume. Elution profiles for the small products obtained from a reaction performed at 30°C are shown in Fig. 4. From this figure it is clear that (a) the average molecular weight is lower than that of the products generated by the enzyme at 0°C, and (b) the percentage of radioactivity present in double-stranded material is smaller than that in the corresponding experiment carried o u t at 0 ° C. We have investigated whether the double-stranded fraction is really a product of the enzymatic degradation or an artifact due to renaturation during the isolation procedure. Small products were isolated, heat denatured and the isolation procedure was repeated to allow eventual renaturation of material to occur. The susceptibility of this material to exonuclease I, which degrades single-stranded DNA to mononucleotides, was compared with that of small products which had not been denatured. Since the formation of acid-soluble products by the enzyme was expected not to give conclusive answers [5], in this case we measured the degradation by means of DEAE-cellulose paper chromatography as described by Davilla et al. [12,13]. By this technique reaction products which differ in size can be selectively eluted from the paper with different buffers. Control experiments showed that native T7 DNA was eluted in Fractions 4 and 5, while the mononucleotides formed by incubation of denatured T7 DNA with exonuclease I were quantitatively recovered in Fraction 1 (Fig. 5a, b). Similarly a large fraction of the small products which eluted in Fraction 3, 4 and 5 was shifted to Fraction 1 after incubation with exonuclease I (Fig. 5c, d). The extent of degradation of the small products obtained with exonuclease I (Table I) indicates that they are 70% single-stranded and that they become 94% single-stranded by denaturation. During the repeated isolation procedure the fraction of material susceptible to exonuclease I decreased to 87% b u t not to 70% as would be the case if the molecules had been renatured completely. Therefore we conclude that the isolation procedure does not allow

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FRACTION NUMBER Fig. 4. S e p h a d e x G - 2 0 0 analysis of t h e p r o d u c t s f r o m d e g r a d a t i o n at 3 0 ° C . (a) U n t r e a t e d small p r o d u c t s . (b) D e n a t u r e d small p r o d u c t s . (c) S m a l l p r o d u c t s i n c u b a t e d w i t h Aspergillus n u c l e a s e $1. See l e g e n d of Fig. 3 f o r f u r t h e r details.

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Fig. 5. E l u t i o n of small p r o d u c t s f r o m D E A E - c e l l u l o s e p a p e r . T h e t e c h n i q u e h a s b e e n d e s c r i b e d b y Davila, Charles a n d L e d o u x [ 1 2 , 1 3 ] . A l i q u o t s of 0.1 m l w e r e s p o t t e d o n W h a t m a n D E A E - c e l l u l o s e p a p e r strips (No. D E S l ) w h i c h w e r e t h e n e l u t e d w i t h t h e f o l l o w i n g solutions: (1) 0 . 0 5 M Tris--HC1 ( p H 7.5). (2) 0 . 0 5 M Tris--HC1 ( p H 7.5), 0 . 1 4 M NaCl. (3) 0 . 0 5 M Tris--HC1 ( p H 7 . 5 ) , 0.5 M NaC1. (4) 0 . 0 5 M T r i s - - H C l ( p H 7.5) 1.0 M NaC1. (5) 1 M N a O H . T h e 3 2 p r a d i o a c t i v i t y in t h e e l u a t e s was d e t e r m i n e d b y C e r e n k o v c o u n t i n g . T h e results are given as t h e p e r c e n t a g e s of t h e t o t a l r a d i o a c t i v i t y e l u t e d . T h e r e c o v e r y of t h e r a d i o a c t i v e m a t e r i a l was c o m p l e t e . (a) N a t i v e T 7 D N A . (b) D e n a t u r e d T 7 D N A a f t e r i n c u b a t i o n w i t h e x o n u c l e a s e I ( 4 8 % acid-soluble m a t e r i a l ) . (c) S m a l l p r o d u c t s u n t r e a t e d . (d) S m a l l p r o d u c t s a f t e r i n c u b a t i o n w i t h 2 u n i t s of e x o n u c l e a s e I p e r #g of D N A f o r 30 rain at 3 7 ° C .

significant renaturation and that the majority, if not all of the double-stranded DNA present after degradation of the DNA by the ATP-dependent DNAase are fragments of the original double-stranded DNA molecules.

Characterization of the intermediates From the results presented in previous paragraphs and from the exonucleolytic character of the degradation it seemed likely that the intermediates contain single-stranded regions at the ends of the molecules. Since it has been shown that the breaks made by the ATP-dependent DNAase are of the 5'P--3'OH type [2] we can determine the length of the single-stranded ends with a 5' terminal by measuring the incorporation of labelled nucleotides with T4 DNA polymerase. The length of the other type (3'OH end) of singlestranded ends can be measured by determining the amount of material which can be made acid soluble with exonuclease I. The results of the experiments with T4 DNA polymerase are shown in TABLE I RENATURATION OF THE SMALL PRODUCTS DURING THE ISOLATION PROCEDURE D e g r a d a t i o n of s m a l l p r o d u c t s b y e x o n u c l e a s e I was m e a s u r e d o n D E A E - c e l l u l o s e p a p e r a n d e x p r e s s e d as p e r c e n t a g e of 3 2 p e l u t e d w i t h B u f f e r 1 (see l e g e n d to Fig. 5). D e n a t u r e d s m a l l p r o d u c t s w e r e o b t a i n e d b y o h e a t i n g f o r 5 rain at 0 C a n d s u b s e q u e n t l y c o o l i n g in ice. Treatment

None Exonuclease I

Percentage of radioactivity eluted f r o m DEAE-cellulose paper with Buffer 1 Small products

Denatured small products

Denatured small p r o d u c t s (reisolated)

0.3 70

0.2 94

2.6 87

174 TABLE

II

INCORPORATION

OF [3H] dTTP IN THE INTERMEDIATES

BY T4 DNA POLYMERASE

T h e i n c o r p o r a t i o n i n t o D N A o f [ 3 H ] d T T P c a t a l y s e d b y T 4 D N A p o l y m e r a s e is e x p r e s s e d a s t h e a m o u n t of acid-insoluble [3HI dTTP. Calf thymus DNA was degraded for about 25% with exonuclease III to serve as a t e m p l a t e f o r t h e p o l y m e r a s e . cpm Incorporated Time (min)

0.86 pg DNA per test

1.0 pg DNA per test

Intermediates

0 10 20

35 388 448

36 557 508

T7 DNA

0 10 20

0 0 103

81 166 45

0 10 20

33 3512 4150

0 3459 3572

Calf Thymus

DNA after EXO III treatment

Table II. The a m o u n t of 3 H incorporated in the intermediates was considerably higher than that found with native T7 DNA. The incorporation of 3H in calf t h y m u s DNA, treated with exonuclease III is a control of the polymerasing system used. From the incorporation of [3 H] dTTP we have calculated that the 5'-terminated single-stranded regions are approx. 0.4% of the intermediates, in other words on the average they are 80 nucleotides long. We could not measure accurately the degradation of the intermediates with exonuclease I due to the low extent of this degradation and the relatively high background value of the control experiment performed w i t h o u t enzyme. F r o m the experiments we could however estimate that the degradation is less than 0.2% of the material. Therefore we tentatively conclude that single-stranded regions with 3'OH are shorter than those at the 5' end or that they are not present at all. Discussion The results presented in this paper indicate that a large fraction of the products formed at an early stage of degradation of T7 DNA by ATP-dependent DNAase from M. luteus, consists of small, dialysable material while the remainder consists of larger molecules which may reach a length of 300 nucleotides. A major fraction of the non-dialysable products is single-stranded, but also molecules have been found, which axe double-stranded or which contain double-stranded regions. These double-stranded regions vary in length between a few nucleotides and 150--300 nucleotides. Friedman and Smith [14] have found that upon degradation of T7 DNA with ATP-dependent DNAase from Haemophilus influenzae double-stranded products are being formed with large single-stranded regions at their ends. In contrast to the M. luteus enzyme, the H. influenzae enzyme releases very little acid soluble material. It has been shown previously that the ATP-dependent DNAases from M.

175

luteus and H. influenzae are exonucleases which operate by binding to the end of a DNA molecule and degrading it to completion before attacking another DNA molecule [3, 15]. Thus products are released while the enzyme is moving along the DNA molecule. In the case of the Haemophilus enzyme, Friedman and Smith [14] have explained the presence of double-stranded molecules with long single-stranded ends by assuming that the enzyme produces "staggered breaks" and melts the region between two breaks so that the products can come apart. Such a mechanism of action does not seem applicable to the M. luteus enzyme since it does not account for the large amounts of acid-soluble material formed by this enzyme. One would have to further postulate that the enzyme rapidly degrades a substantial fraction of the single-stranded ends into oligonucleotides of variable length. The enzyme however, has been shown to posess very little activity towards single-stranded DNA. The presence of acid soluble material, besides relatively long single-stranded molecules and/or double-stranded molecules with long single-stranded ends, would rather suggest a mechanism by which the enzyme nicks one strand every few hundred nucleotides, and the complementary strand every few nucleotides. When the distance between t w o breaks is sufficiently short the oligonucleotides will melt off, leaving single-stranded regions [16]. If, however, the distance between breaks is larger than 10 nucleotides a region of double-stranded DNA will be preserved. Our data do not allow us to conclude, whether the long single-stranded products originate from a particular strand. The presence of long singlestranded regions with 5' termini and the apparent absence of single-stranded regions with opposite polarity at the ends of the intermediates of high-molecular weight suggest that the enzyme specifically forms long single-stranded products from the strand with 5'--3' polarity. Such a conclusion, however, has to be drawn with some reservations, because of the uncertainties in the determination of single-stranded regions with 3' termini. Experiments with DNA labelled with different radioisotopes at the 5' and 3' ends of the molecules might resolve this problem. Such experiments are at present being carried out in this laboratory.

Acknowledgements We thank H.L. Heijneker for a gift of T4 DNA polymerase, exonuclease I and exonuclease III. We gratefully acknowledge the help of R.A. Oosterbaan and S. Bacchetti in the preparation of the manuscript. This work was supported by the Euratom contract 052--65--2 BIAN. References 1 Hout, A., Oosterbaan, R.A., Pouwels, P.H. and de Jonge, A.J.R. (1970) Biochim. Biophys. Acta 204, 632--635 2 Anal, M., Hirahashi, T. and Takagi, Y. (1970) J. Biol. Chem., 245, 767--774 3 V a n Dorp, B., Ceulen, M. Th.E., Heyneker, H.L. and Pouwels, P.H. (1973) Biochim. Biophys. Acta 299, 65--81 4 Takagi, Y., Matsubara, K. and Ansi, M. (1972) Biochim. Biophys. Acta 269, 347--353 5 Cleaver, J.E. and Boyer, H.W. (1972) Biochim. Biophys. Acta 262, 116--124 6 Furlong, N.B. Methods in Enzymology (Moldave, K. and Grossman, L., eds), Vol. XII A, pp. 320--321, Academic Press, N e w York, L o n d o n 7 Lehman, I.R, and Nussbaum, A.L. (1964) J. Biol. Chem. 2628--2636

176 8 9 10 11 12 13 14 15 16

V o g t , V.M. ( 1 9 7 3 ) E u r . J. B i o c h e m 33, 1 9 2 - - 2 0 0 G o u l i a n , M., L u c a s , Z0J. a n d K o r n b e r g , A. ( 1 9 6 8 ) J, Biol. C h e m . 2 4 3 , 6 2 7 - - 6 3 8 N y g a a r d , A.P. a n d H a l l , B.D. ( 1 9 6 4 ) J. Mol. Biol. 9, 1 2 5 - - 1 4 2 S t u d i e r , T.W. ( 1 9 6 5 ) J . Mol, Biol. 11, 3 7 3 - - 3 9 0 Davila, C., C h a r l e s , P. a n d L e d o u x , L. ( 1 9 6 5 ) J. C h r o m a t o g r . 1 9 , 3 8 2 - - 3 9 5 Davila, C., C h a r l e s , P. a n d L e d o u x , L. ( 1 9 6 5 ) J. C h r o m a t o g r . 19, 3 9 6 - - 4 0 3 F r i e d m a n , E . A . a n d S m i t h , H . O . ( 1 9 7 3 ) N a t . N e w Biol. 2 4 1 , 5 4 - - 5 8 F r i e d m a n , E . A . a n d S m i t h , H . O . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 2 8 4 6 - - 2 8 5 3 W a n g , J.C. a n d D a v i d s o n , N.J. ( 1 9 6 6 ) J. Mol. Biol. 15, 1 1 1 - - 1 2 3