12 Deoxyribonuclease I

Deoxyribonuclease I M. LASKOWSKI, SR. I. Introduction . 11. Chemical Nature 111. Active Center . IV. Inhibitor . . V.Ions . . . VI. Kinetics . . VII. Specificity . . VIII. Physiological Role

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289 292

297 299 302 303 308 310

I. Introduction

During the past decade a previously accepted notion that the deoxyribonucleic acid (DNA)-deoxyribonuclease (DNase I) reaction runs a uniform course with a uniform specificity began to be seriously doubted. It is now realized that striking differences exist between the early and terminal stages of the same reaction. The observed differences are not limited solely to the rate of the reaction but include variations in endoor exonucleolytic character, in the effect of the divalent cation, and, finally, in the specificity toward the bases adjacent to the bond that is cleaved. Several reviews and books devoted exclusively t o methodology for following the action of DNase I exist (1-5). For the purpose of this 1. “Methods in Enzymology,” Vol. 2, Sect. 2, 1955. 2. “Methods in Enzymology,” Vol. 6, Sects. 1 and 2, 1963. 3. “Methods in Enzymology,” Vol. 12, Parts A and B, 1967, 1968. 4. G. L. Cantoni and D. R. Davies, Procedures Nucleic Acid Res. Sect. A (1966). 5. N. Kurnick, Methods Biochem. Anal. 9, 1 (1962). 289

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M. LASKOWSKI, SR.

review it suffices to say that it is possible by the use of the p H stat to measure the number of internucleotide bonds cleaved. This method is independent of the stage of the reaction or of the location of the bond within the molecule. Methods measuring changes in molecular weight reflect the number of double-strand scissions. Finally, with the use of two other enzymes, polynucleotide kinase and DNA ligase (see below), it is possible to evaluate the number of “nicks” inflicted on one of the strands without causing a scission. Methods based on spectrophotometry reflect the collapse of ordered structure. Finally, methods measuring the appearance of mononucleotides reflect exonucleolytic activity. Among endonucleases which hydrolyze DNA one seldom finds an enzyme that attacks double-stranded and single-stranded substrates with equal ease. If the enzyme shows preference for double-stranded substrates (as DNase I does) autoretardation is observed. This decrease in the reaction rate is caused by the gradual disappearance of the preferred, double-stranded substrate and an increase in the concentration of less susceptible, single-stranded substrate. Differences in rates between the early and terminal phases of the reaction of the order of 1OOOfold have been described ( 6 ) . The opposite case, autoacceleration, is seen with those enzymes that show preference for the single-stranded structure, e.g., micrococcal nuclease ( 7 ) . I n the original meaning (8,9)endonucleases and exonucleases were conceived as retaining their character throughout the whole course of the reaction. It is now established that at least some typical endonucleases acquire exonucleolytic character toward the end of the reaction (10). Proximity of the newly created monophosphoryl or hydroxyl group is responsible for this change. Many DNases are known to be activated by a divalent cation. However, only from the work of Bollum (11) did it become clear that the nature of the cation may qualitatively change the specificity of the enzyme toward adjacent bases. Quantitative changes in the requirements for the divalent cation (10) have been observed during different stages of the same reaction, e.g., micrococcal nuclease (7) where the increased Ca2+concentration causes a decrease in the average size of the terminal product. Finally, it was shown with a number of DNases that during the 6. S. Vanecko and M. Laskowski, Sr., JBC 236, 3312 (1961).

7. E.Sulkowski and M. Laskowski, Sr., JBC 243, 4917 (1968). 8. M.Privat de Garilhe and M. Laskowski, Sr., JBC 223, 661 (1956). 9. M.Laskowski, Sr., G. Hagerty, and U.-R. Laurila, Nature 180, 1181 (1957) 10. M. Laskowski, Sr., Advan. Enzymol. 29, 165 (1967). 11. F.J. Bollum, JBC 240, 2599 (1965).

12. DEOXYRIBONUCLEASE

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291

course of the reaction cleavages become less specific (10). By extrapolation, the first few cleavages must be very specific. The direct proof for this statement is delayed by technical difficulties in determining terminal nucleotides in fragments of 1000 or more monomers. I n spite of this, it seems safe to predict that in the near future the DNases previously considered to be nonspecific will be used to inflict a very limited number of very specific cleavages. Regulation of ionic medium, pH, temperature, and exposure time can be expected to significantly improve specificity in the early stages of the reaction. On the other hand, it becomes equally evident that in the terminal stages of the reaction, in addition to the base specificity the effect of the monophosphoryl group determines the end point of the reaction (1.2). The reasons for selecting pancreatic DNase I as one of the two representative of mammalian DNases are to a large extent historical. Deoxyribonuclease I was the first enzyme to be recognized as specific for DNA (13-16),the first DNase to produce 5’-monoesterified products (16, lY), the first DNase to be crystallized (It?), the first DNase to have a specific protein inhibitor (19-23),the first DNase shown to produce “nicks” on one strand in preference to scission of both strands ( 2 4 , 2 6 ) .A new first has been added recently (25a) ; DNase I was covalently coupled to porous glass, thus supplying an insoluble DNase. The articles on DNases in previous editions of “The Enzymes” (26,xT) discussed several of these issues in historical perspective. The historical discussion will not be repeated in the present edition except when new information requires an introduction. 12. A. J. Mikulski, E. Sulkowski, L. Stasiuk, and M. Laskowski, Sr., JBC 244, 6559 (1969). 13. J. P. Greenstein and W. V. Jenrette, J . Natl. Cancer Inst. 1, 845 (1941). 14. M. Laskowski, Sr. and M. K. Seidel, A B B 7, 465 (1945). 15. M. McCarty, J . Gen. Physiol. 29, 123 (1946). 16. J. L. Potter, K. D. Brown, and M. Laskowski, Sr., BBA 9, 150 (1952). 17. R. L. Sinsheimer and J. F. Koerner, JACS 74, 283 (1952). 18. M. Kunitz, J . Gen. Physiol. 33, 349 (1950). 19. W. Dabrowska, E. J. Cooper, and M. Laskowski, Sr., JBC 177, 991 (1945). 20. E. J. Cooper, M. L. Trautman, and M. Laskowski, Sr., Proc. SOC. Exptl. B i d . M e d . 73, 219 (1950). 21. L. Cunningham and M. Laskowski, Sr., BBA 11, 590 (1953). 22. U. Lindberg, Biochemistry 6, 323 (1967). 23. U. Lindberg, Biochemistry 6, 343 (1967). 24. S. Zamenhof, G. Griboff, and N. Marullo, BBA 13, 459 (1954). 25. E. T. Young, I1 and R. L. Sinsheimer, JBC 240, 1274 (1965). 25a. A. R. Neurath and H. H. Weetall, FEBS Letters 8, 253 (1970). 26. M. Laskowski, Sr., “The Enzymes,” 1st ed., Val. 1, p. 956, 1951. 27. M. Laskowski, Sr., “The Enzymes,” 2nd ed., Val. 5, p. 123, 1961.

M. LASKOWSKI, SR.

II. Chemical Nature

Almost as soon as bovine pancreatic crystalline DNase I was obtained, doubts concerning its homogeneity arose. Even before crystallization (18) it was shown (28,282sa) that DNase I cocrystallizes with chymotrypsinogen B. The chronologically first crystalline product contained about two-thirds chymotrypsinogen B and about one-third DNase I (28, 28a). It would, therefore, be expected that the reverse also occurs. I n fact, Potter (29) showed that a commercial sample of crystalline DNase can be separated into five protein-containing bands on cellulose acetate strips. One of these bands was identified as chymotrypsinogen B. With a sensitive method of detection (31) the presence of one part of RNase per 100,000 parts of DNase I was found in the crystals. This activity could be further reduced by continuous flow electrophoresis (S2), or more efficiently by chromatography on DEAE-cellulose (33). Lindberg (34) passed the solution of commercial crystalline DNase I through a column of Sephadex G-100, removed contaminants of smaller molecular weight, and obtained a preparation of high purity. A series of beautiful papers from the laboratory of Moore and Stein ( 3 5 3 9 a ) recently appeared, confirming that crystalline DNase I is contaminated with about one-third chymotrypsinogen B and chymotrypsin B. It was first established (35) that commercial DP grade ( 3 2 ) DNase I is composed of a t least two, enzymically active glycoproteins. Figure 1, reproduced from the work of Price et al. ( S 5 ) , shows the separation on SE-Sephadex into two equally active components B and A. A modified procedure (39) of chromatography on phosphocellulose (Fig. 2) led to three peaks, C, B, and A (in order of their appearance from the column). A further refinement of technique (39a) allowed visualization of a small additional 28. M. Laskowski, Sr., JBC 166, 555 (1946). 28a. M. Laskowski, Sr. and A. Kazenko, JBC 167, 617 (1947). 29. J. L. Potter, personal communication, quoted by Laskowski (SO). 30. M. Laskowski, Sr., Procedures Nucleic Acid Res. p. 83 (1966). 31. J. Polatnick and H. L. Bachrach, Anal. Biochem. 2, 161 (1961). 32. Worthington, catalog (1965). 33. S. B. Zimmerman and G. Sandeen, Anal. Bioehem. 14, 269 (1966). 34. U. Lindberg, Biochemistry 6, 335 (1967). 35. P. A. Price, T.-Y. Liu, W. H. Stein, and S. Moore, JBC 244, 917 (1969). 36. P. A. Price, S. Moore, and W. H. Stein, JBC 244, 924 (1969). 37. P. A. Price, W. H. Stein, and S. Moore, JBC 244, 929 (1969). 38. B. J. Catley, S. Moore, and W. H. Stein, JBC 244, 933 (1969). 39. J. Salnikow, W. H. Stein, and S. Moore, Federation Proc. 28, 344 (1969). 398. J. Salnikow, S. Moore, and W. H. Stein, JBC 245, 5685 (1970).

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peak D. Each component had comparable specific activity. Peaks A:B:C were present in a constant ratio of 4:l:l. The major peak A corresponded to peak A of the previous separation (35). Figure 2 shows a portion of the chromatographic pattern obtained on phosphocellulose. Peak A has been studied further. Table I, reproduced from the paper of Price et al. (35),compares amino acid content of peak A with that obtained by Lindberg (34) for the purified DNase I. The composition reported from the two laboratories for preparations obtained by two different methods is surprisingly similar. The linear sequence is being determined in the laboratory of Moore and Stein (39b). The possibility that the three fractions are artifacts caused by the exposure to a strong acid during the preparation procedure has been ruled out by the experiment with a freshly collected pancreatic juice. The juice was first chromatographed on DEAE-cellulose according to Keller et al. (40) and the fraction containing DNase was then rechromatographed on phosphocellulose (39) and gave three identical peaks as were seen with crystalline DNase. To elucidate the mode of attachment of the carbohydrate moiety to the protein of DNase I, Catley et al. (38)digested the peak A DNase with Pronase and subjected the digest to gel filtration on Sephadex G-25. All of the carbohydrate was recovered in a mixture of the dipeptide SerAsp and the tetrapeptide Ser-Asp-Ala-Thr. Removal of serine by an Edman degradation demonstrated that all of the carbohydrate was in association with aspartic acid. Analysis of the carbohydrate moiety demonstrated two residues of glucosamine, five residues of mannose, and one residue of ammonia, leading to the conclusion that the saccharide moiety is attached at a single position on the enzyme through an aspartamidohexose linkage. An analysis of the peak B DNase identified the same sugars in the same proportions except that sialic acid was present in fractional quantities. Since sialic acid in the intact peak B DNase analyzed for 0.2 residue and in a tetrapeptide fraction for 0.06 residue, it was considered to be part of an impurity not associated with the heptasaccharide moiety. If the presence of fractional quantities of sialic acid is accidental, the most probable reason for the separation of peaks C, B, and A appears to be the number of amides since the gross amino acid composition is not different. Some minor corrections of the values shown in Table I are required. The latest work (39a) shows that peaks A and B have identical amino acid composition. Peak A contains two residues of N-acetylglucosamine 39b. S. Moore and W. H. Stein, personal communication (1970). 40. P. J. Keller, E. Cohen, and H. Neurath, JBC 233, 344 (1958).

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FIG.1. Chromatography of various samples of DNase on SE-Sephadex column, 0.9 by 55 cm; temperature, 25" ; initial eluent 0.2 M sodium acetate buffer, p H 4.70; linear gradient with 150 ml each of the initial and limit buffer, 1.OM sodium

Enzymic activity. (A) Worthington D P grade DNase acetate buffer, pH 4.70. (0) (amorphous), load 30 mg, linear gradient begun a t 30 ml. (B) Worthington oncecrystallized DNase, load 45 mg, linear gradient begun a t 21 ml. (C) Worthington electrophoretically purified DNase, load 5 mg, linear gradient begun a t 20 ml. From Price et al. (36). Authentic sample of chymotrypsinogen B tested in the same system was eluted between 10 and 35 effluent ml. Chymotrypsin B gave several peaks in the range 15-65 ml.

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FIG. 2. Chromatography of DNase I (Worthington D P grade) on phosphocellulose (Whatman P-11, column 0.9 by 70 cm) linear gradient of sodium acetate buffer pH 4.7, 0.38-0.7 M (with respect to acetate), 200 ml of each. From Salnikow et al. (39).

296

M. LASKOWSKI, SR.

TABLE I AMINOACID COMPOSITION OF PANCREATIC DEOXYRIBONUCLEASE PEAKA a - b ~~

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Nearest integral number of residues per molecule Residue

Residues per 100 g of protein

Residues Per molecule

Price et al. (Ref. 56)

3.78 2.65 6.08 12.08 4.88 8.41 8.37 2.93 1.75 5.13 8.55 4.33 8.49 8.30 5.47 1.71 1.31 2.34 0.97 0.95

9.14 5.99 12.07 32.81 14.96 29.92 20.00 9.35 9.51 22.37 26.72 11.86 23.26 15.77 11.52 4.05 3.95 3.90 1.82 1.81

9 6 12 33 15 30 20 9 10 22 27 12 23 16 12 4 4 4 2 2

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Lysine Histidine Arginine Aspartic acid Threoninec SerineC Glutamic acid Proline Glycine Alanine Valined Isoleucined Leucine Tyrosinec Phenylalanine Methioninea Half-cystinee Tryptophan/ Mannose Glucosamine Amide-NH3 Total

Lindherg (Ref. 34) ~~

9 6 12

34 15 30 20 9 9 23 27 12 24 16 12 4 4 4 22

98.589

272

From Price et al. (36). Duplicate analyses by ion exchange chromatography were performed on 24-, 48-, and 72-hr hydrolyzates. Unless indicated otherwise, the average of all of these values was used to compute the values in the second column. Replicate analyses agreed to *2.5y0. A molecular weight of 31,000 was used to calculate the residues per molecule. The average 24-, 48-, and 72-hr values for threonine, serine, and tyrosine have been extrapolated to zero time to correct for destruction during hydrolysis. The average 72-hr values were taken for isoleucine and valine. Methionine and half-cystine were determined in duplicate as methionine sulfone and cysteic acid on the performic acid-oxidized protein [S. Moore, JBC 238, 235 (1963)l. A molecular ratio of 3.80 was determined for tyrosine to tryptophan by the method of T. W. Goodwin and R. A. Morton [BJ 40, 628 (1946)l. g This recovery figure is based on the weight of the samples analyzed after correction for moisture and ash content.

12.

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and six residues of mannose. Peak B contains one residue of sialic acid, three residues of N-acetylglucosamine and five residues of mannose. Peak C contains the same carbohydrate as peak A, but one residue less of histidine and one residue more of proline. All three forms have NH, terminal leucine, COOH-terminal threonine. Other changes from the values of Table I are in tryptophan, three rather than four residues, aspartic acid 34 rather than 33, and valine 25 rather than 27. The amino acid analysis (Table I) strongly supports the value of 31,000 for the molecular weight of DNase I. This value has been accepted in Stockholm (34) and in New York (35), making all previously reported values obsolete [see reviews (10, 26, 27, SO)1. The values obtained (Table 11) by ultracentrifugation methods sedimentation diffusion and approach to equilibrium closely agree with the value of 31,000 based on N-terminal determination. New information is also available (35) on stabilization of the DNase I molecule. The presence of 5 mM Caz+fully stabilizes the molecule against proteolytic digestion. For many years it has been known that pancreatic proteases, trypsin and both chymotrypsins, are protected from autolysis by 10mM Ca2+.The protection of DNase I is achieved by a lower Ca2+ concentration. Calcium also exerts a pronounced effect on the reduction of S-S bonds in DNase I. This protein is unusually susceptible to reduction: Both S-S bonds may be reduced within minutes by mercaptoethanol or similar reagents a t pH 7.2 without any denaturing agent (37). The reduced enzyme is inactive and remains inactive even after exposure for 24 hr to oxygen in the presence of ethylenediaminetetraacetate (EDTA) . The situation changes upon addition of 4 m M Ca2+.Activity is regained in minutes. If the Ca2+is present during reduction, only one S-S bond is reduced and activity remains unchanged. If Ca2+is added to a completely reduced protein even in the presence of a reducing agent one S-S bond is re-formed. In spite of exerting a strong stabilizing effect on the enzyme, Ca2+is not strongly bound to the protein. Simple gel filtration a t neutral pH removed .l6Ca2+completely from both the native and the partially reduced forms of the enzyme (37).

111. Active Center

Neither the three-dimensional structure nor the complete primary structure of DNase I has yet been announced. However, a histidine residue has been identified in the active center of DNase I fraction A by

298

M. LASKOWSK1, SR.

SUMMARY OF

THE

TABLE I1 PHYSICAL AND CHEMICAL CHARACTERISTICS OF DNase 1.

Characteristic analyzed

Ei:m a t 280 nm Experimental From amino acid composition Refractive index increment (ml/g)' szO,a ( X 10-13 sec) D20,w( X 10+ cm2/sec) Partial specific volume (from amino acid composition)

Results obtained 12.3b 13.9" 15.3d 0.196 f 0.007f 2.78 8.7 0.733

Molecular weight Approach to equilibrium Sedimentation diffusion N-Terminal determination Isoelectric point Experimental Total nitrogen (%) Experimental From amino acid composition Total sulfur (yo) Experimental From amino acid composition ~~

30 ,900 30,700 29 ,400 31,300 4.79 16.7 f 0.4f 16.2 0.75 f 0.02f 0.81

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Reprinted from Lindberg (34) by permission of the copyright owner. Copyright 1967 by the American Chemical Society. b In 0.01 M KzHP04-KH2P04buffer, pH 7.6. I n 0.1 N NaOH, pH 13. d This value was calculated using the molar extinction coefficients for the aromatic amino acids tyrosine and tryptophan given by T. W. Goodwin and R. A. Morton [BJ 40, 628 (1946)). 8 Here dn/dc was determined at 20", using the ultracentrifuge as a differential refractometer. f The average deviation about the mean is given based on four determinations. 0 Determined by M. Kunitz [ J . Cen. Physiol. 33, 349 (1950)] and by A. Polson [BBA 22, 61 (1956)j. 0

Price et al. (36).The evidence is based on experiments in which DNase I was reacted with iodoacetate a t pH 7.2 in the presence of 0.1 M Mn2+. Under these conditions the enzyme is gradually inactivated and the loss of activity parallels the formation of one residue of 3-carboxymethyl histidine per molecule. The rate of the alkylation reaction is dependent on Mnz+concentration. Substitution of Mn2+by Cuz+in the presence of tris buffer greatly increases the rate of alkylation. A 29-residue peptide con-

12.

DEOXYRIBONUCLEASE I

299

taining the modified histidine residue has been isolated in 90% yield after tryptic hydrolysis of carboxymethylated DNase I. One structural requirement in the active center can be deduced from a study of specificity with small substrates (see Section VII). Oligonucleotides bearing a 3'-monophosphoryl group are easily cleaved to form d-pNwp (41), whereas neither N u nor pN@is formed. This suggests that a positively charged group (either from lysine or arginine) is placed in the vicinity of an active histidine and immobilizes the negative charge of 3'-monophosphoryl group. One can further speculate that the resistance of d-N"pN0 and d-pN"pN@to the action of DNase I is caused by the lack of such an anchor. However the resistance of a t least some compounds of the type d-pN"pN@cannot be explained without postulating the repulsive effect of the 5'-monophosphoryl group. The long-established preference for the Pu-pPy bond is presumably of lesser structural importance than the phosphoryl group because d-ApApTp can be cleaved to liberate d-pTp (@), whereas d-CpApC is resistant (43). IV. Inhibitor

A naturally occurring inhibitor of DNase I was originally observed in hypertrophic epithelium of the crop gland of a pigeon (19) and later in several normal and neoplastic mammalian tissues (20, 2 1 ) . The early work has been reviewed (10). For several years, except for sporadic confirmation, the problem remained dormant until Lindberg (22, 23, 4.4, 45) resumed the work and purified two different proteins with inhibitory properties from calf spleen. The spleen inhibitor I1 was crystallized. It forms a uni-uni molecular complex with DNase I under conditions in which either the inhibitor or DNase I is in excess. Figure 3, reproduced from Lindberg's paper (23), illustrates this point. The complex can be irreversibly dissociated (with the loss of inhibitory activity) into its component parts a t pH 7.6 in the presence of 3 M urea. It can also be dissociated a t pH 11.3 and a t pH 3.5 again with loss of inhibitor activity, the activity of DNase I remaining intact. Recently, Lindberg and Skoog (45a) purified DNase I inhibitor from thymus. A 15-fold purification led to a homogeneous preparation. The thymus inhibitor has many properties identical with spleen inhibitor I1 but differs in molecular weight 41. S. Vanecko and M. Laskowski, Sr.,JBC 236, 1135 (1961). 42. J. L. Potter, U.-R. Laurila, and M. Laskowski, Sr.,JBC 233, 915 (1958). 43. H. G. Khorana, J . Cellular C o m p . Physiol. 54, Suppl. 1, 5 (1959). 44. U. Lindberg, BJ 92, 27p (1964). 45a. U. Lindberg and L. Skoog, European J . Biochetn. 13, 326 (1970).

300

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FIG.3. Sephadex G-100 chromatography on DNase I, inhibitor 11, and mixture containing the two proteins. (A) DNase I only, (B) inhibitor I1 only, (C) and (D) both components with different molar excess of inhibitor, (E) equimolar amounts of inhibitor and enzyme, (F) and ( G ) both components with a different excess of enzyme. Absorbance a t 215 nm (solid line) was measured after 20-fold dilution with water using a similarly diluted blank of the elution buffer (0.5 M potassium phosphate, p H 7.6). Each chromatogram was analyzed for DNase activity (01, inhibitor activity ( O ) , and for the presence of DNase-inhibitor complex, in this figure represented as DNase I activity which was measured on samples of the fractions after adjustment of the pH to 3.5 with HCl ( 0 ) .[From Lindberg (3 4 ). Copyright 1967 by the American Chemical Society. Reprinted by permission of the copyright owner.]

12. DEOXYRIBONUCLEASE I

30 1

(49,000 instead of 59,000 for spleen inhibitor 11) and in the maximum stability which is a t pH 6 for thymus and pH 7 for spleen. One of the most exciting aspects of the problem is the exact mechanism of the union of the two proteins. Striking progress in understanding of the mechanism of the union between trypsin and trypsin inhibitor has been made in the laboratory of Laskowski, Jr. (46, 4 7 ) . The trypsin inhibitor-trypsin complex is essentially a Michaelis-Menten complex. During the union one trypsin-sensitive bond in the inhibitor (reactive site) is cleaved without affecting the inhibitory power of the protein, thus creating “the modified form” of the inhibitor. If one makes an analogy to enzyme other than trypsin, it would be expected that the inhibitor for DNase I should be a specifically resistant DNA. However, the protein nature of both partners, DNase I and spleen inhibitor 11, is well established. Obviously, not all protein-protein interactions must be of the trypsin inhibitor-trypsin type, but it may be worthwhile to check the DNase inhibitor for possible proteolytic activity. One is tempted to speculate that in this case the inhibitor I1 may be a protease specific for a peptide linkage involving histidine. To prove it experimentally, it would be necessary to trap the “modified form of DNase I” before the bond is re-formed. Several years ago the question of whether DNase I is a strictly digestive enzyme was argued. On the basis of experiments in which DNase I type of activity was found in the minced tissue only after the previous exposure to an acid pH, known to destroy the inhibitor, it was concluded (21) that the DNase I type of enzyme is intracellular and ubiquitous. Recently, Lee and Zbarsky (48) used pigeon crop gland inhibitor to identily a DNase I type of activity in the intestional mucosa of the rat. The crop gland inhibitor and presumably the spleen inhibitor I1 react with DNase I of many species. It appears that no species specificity exists in the reaction of complex formation. The above considerations could bring us to the discussion of the occurrence of DNase I in different tissues and in different organelles of the same cell. A fairly extensive literature exists, particularly in reference to different pathological conditions. It will not be considered in this review. In many papers only two criteria are used to classify an enzyme as DNase I : (1) It requires Mg”, and (2) it has an optimum a t pH 7. 46. M. Laskowski, Jr., S y m p . , Structure-Pri?iction Relationship of Proteolytic En(P. Desnuelle, H. Neurath, and M. Ottessn, eds.) p. 89. Munksgaard, Copmliagen, and Academic Press, New York. 1970. 47. M. Laskowski, Jr. and R . W. Senlock, “The Enzymes,” 3rd ed., Vol. 111, p. 376, 1971. 18. C. Y. Lee and S. 11. Zbarsky, Can. J . Biochem. 45, 39 (1967). zymes, 1968

302

M. LASKOWSICI, SR.

Historically, these criteria are justified, but they are no longer sufficient. Additional information characterizing the enzyme as a 5'-monoester former would be desirable, but the ability to react with the specific DNase I inhibitor appears to be the most important criterion. Without having tested for it the enzyme should not be called DNase I.

V. Ions

Many years ago it was observed that the presence of either Mg2+or MnZ+increased the rate of hydrolysis of DNA by DNase I whereas high concentrations of NaCl decreased it [see review (10)1. The revolutionary finding comes from the work of Bollum (11) who showed that the nature of activating cation qualitatively affects specificity. Deoxyribonuclease I was presented with (dI),.(dC), as substrate. Only (dI), but no (dC), was hydrolyzed when 1 0 m M Mg2+ was the sole activating divalent cation. If, in addition to Mg2+,2 mM Ca2+was introduced, both strands were hydrolyzed. The same result (both strands digested) was obtained when 10 m M Mn2+ alone was present instead of the mixture of Mg2+and Ca2+.A recent paper (48a) describes the elegant separation and identification of di- and trinucleotides in the DNase I digest. The composition of digests obtained in the presence of Mn2+is different from that obtained in the presence of MgZ+. This result strongly supports Bollurn's (11) conclusion. This discovery of Bollum (11) makes obsolete a number of previous excellent studies including those on mutual interdependence between concentrations of divalent metal, monovalent metal, hydrogen ion, and substrate. Unless the bond affected by the metal in question is specified, an overall rate represents a number with little value. The problem is further complicated by the suspected (by analogy to other nucleases) quantitative changes in requirements for metal ions a t different stages of the reaction. So far no such data are available for DNase I. One is tempted to add, luckily, because in view of the uncertainty of qualitative effects such data would hardly be expected to have a long survival time. Melgar and Goldthwait (49, 50) used a method in which isotopically labeled DNA was incorporated into acrylamide gel. The suspension of the gel containing DNA was used as substrate. The average molecular 48a. E. Juchnowicz and J. H. Spencer, Biochemistry 9, 3640 (1970). 49. E. Melgar and D. A . Goldthwait, JBC 243, 4401 (1968). 50. E. Melgar and D. A. Goldthwait, JBC 243, 4409 (1968).

12.

DEQXYRIBONUCLEASE I

303

weight of fragments released from the gel was approximately 400,000. In the presence of Mg2+ alone the rate of release of these fragments with DNase I showed a lag period. No such lag period was observed with either DNase I1 or E . coli endonuclease I, known to make doublestrand scissions. The lag was eliminated when Mn2+,Caz+, or Co2+,or Mg2+plus Ca*+was used. Sodium added to Mn2+,to Ca2+,or to Ca2+plus Mg2+reestablished the lag. The results are interpreted as indicating that only single-strand cleavages occur during the lag period, whereas double-strand scissions release the fragments from the gel. This interpretation was confirmed by viscometry and ultracentrifugation. Eichhorn et al. (51) concluded that Co*+is a better activator of DNase I than any of the previously used metals. As a criterion of activity the authors used the formation of acid-soluble products, corresponding to terminal stages of the reaction. I n neither of these papers (49, 51) was the attempt made to characterize the split bond. VI. Kinetics

As mentioned in the Introduction, a characteristic aspect of the kinetics of DNase I acting on native DNA is autoretardation (10). Autoretardation is caused by the continuous formation of products which are poorer substrates than those from which they are derived. Three types of experiments were performed to prove this (6). Experiments of the first type were performed in a p H stat. The reaction was allowed to run until the originally fast rate reached a plateau. At this time a 100-fold excess of enzyme was added. The initial rate of the reaction was restored, then slowed down, and reached a second plateau. Further addition of a fourfold amount of enzyme (the total enzyme concentration was now 500-fold that of the original) resulted in a new burst of activity, but the rate was slower than the original. A second type of experiment was performed by isolating the reaction products a t different stages of the degradation and using them as substrate for a fresh sample of DNase I. The reaction again was followed in a p H stat. The rate was highest with native DNA. To obtain comparable rates the amount of enzyme needed was 15-fold with the “I-min digest,” 500-fold with the “10-min digest,” and 2500-fold with “oligonucleotides” obtained from the reaction in which the first plateau was reached. 51. G. L. Eichhorn, P. Clark, and E. Tarien, JBC 244, 937 (1969).

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The third type of experiment was performed to show that inhibition by the products is insufficient to account for the observed difference in rate. The substrate consisted of a 50:50 mixture (in terms of optical density) of native DNA and “oligonucleotides.” With this mixture the rate of hydrolysis decreased to about one-half of that for native DNA. This result agrees with the previously established (52) competitive-type inhibition by the products. It also shows that the inhibition by products accounts for changes in rate within one order of magnitude, whereas the decreasing affinity toward the newly formed substrates accounts for more than three orders of magnitude. With so pronounced a difference in susceptibility of substrate in the very early and in the very late phases of the reaction, the two phases must be considered independently. Kinetics of the early phases of the reaction have been studied by Dekker and Schachman (53),Schumaker et al. (54),Thomas ( 5 5 ) , and Young and Sinsheimer ( 2 5 ) . All the results demonstrate that DNase I hits a t random and that several hits on a single strand occur before one double-strand scission is detected. The estimates of numbers, however, vary; Thomas considered that an average of 200 single hits occur before the average molecular weight has decreased by a factor of 2. Young and Sinsheimer (25) estimated that an average of 4 hits occur before h-phage DNA is inactivated, possibly as a result of a doublestrand scission. The ability of DNase I to inflict a number of a single-strand breaks (or nicks) before producing a detectable decrease in molecular weight has been utilized by Richardson and his colleagues to create a detecting system in their studies of polynucleotide kinase and DNA ligase (56, 5 t h ) . The scheme of sequential reactions involved in nicking and labeling the nicked ends is reproduced in Fig. 4. Figure 5 shows the dependence between the number of single-strand breaks and concentration of DNase I. In view of the impact of this work on several lines of nucleic acid research some of the experimental details are given in the legends. Figure 6 shows the scheme of a procedure by which the labeled nicks are first closed with the aid of DNA ligase. Deoxyribonucleic acid is then degraded enzymically in such a manner that allows the identification of the nucleosides adjacent to the labeled internucleotide linkage. Table I11 shows the composition of nucleotides adjacent to the nick 52. L. F. Cavalieri and B. Hatch, JACS 75, 1110 (1953). 53. C. A. Dekker and H. K. Schachman, Proc. Natl. Acad. Sci. U . S. 40,894 (1954). 54. V. N. Schumaker, E. G. Richards, and H. K. Schachman, JACS 78,4230 (1956). 55. C. A. Thomas, Jr., JACS 78, 1861 (1956). 56. B. Weiss, T. R. Live, and C. C. Richardson, JBC 243, 4530 (1968). 56a. B. Weiss, A. JaqueminSablon, T. R. Live, G. C. Fareed, and C. C. Richardson, JBC 243, 4543 (1968).

32PJ-

L-

-P -"J

1

Polynucleotide Kinase APP+p3'

P32

.-I

PY

FIG.4. Scheme of the preparation containing =P-labeled phosphomonoesters a t single-strand breaks, The two strands of TT DNA duplex are schematically represented by two parallel lines and only the 5' termini are designated. After the introduction of single-strand breaks into DNA by incubation with pancreatic DNase, the phosphomonoesters formed are removed by phosphatase a t 65". The 5' termini are then labeled by incubation with polynucleotide kinase. From Weiss et al. (66).

DNase concentralion,units /ml

1 k . 5. Production of single-strand breaks by pancreatic DNase. T, DNA w m incubated with varying amounts of pancreatic DNase. After each reaction, the number of single-strand breaks (internal phosphomonoesters per 4.0 X 10' nucleotides) was measured by end group labeling (see Fig. 4 ) . Crystalline pancreatic DNase I (11 mg. 1 vial Worthington) was dissolved in a 1-ml solution containing 10 m M sodium acetate buffer, pH 5.5,5 mM MgCl,, 0.2M NaC1, and 0.5 mg/ml of bovine plasma albumin. The mixture was stored at 0" for up to 1 month during which time it gradually lost 10-25% of its activity. Immediately before use it was diluted with the same solution and assayed spectrophotometrically by the method of Kunitz. One unit of enzymic activity was defined as the amount of enzyme causing an increase in Az, of 0.001/min/ml of assay solution at 25". Deoxyribonucleic acid was incubated in 5 ml volume containing 1.3 mM DNA, 67 mM tris-HC1 buffer (pH 8.0), 5 m M MgCl,, and 0.5-5 units of DNase I at 20" for 30 min. To stop the reaction EDTA (0.5M,pH 7.5) was added to attain 16 mM concentration. The niixture was dialyzed for 8 hr at 4" against 20 n d 1 NaCl-10 mM tris-HC1 buffer (pH 8.0) and stored up to 6 months at 0".From Weisa et al. (66).

306

M. LASKOWSKI, SR. X

Y

Z

A B C

- -- p l p J p .bz i p l

p

1,-- -

I

Ligose system

X Y Z A B C

- - -pJ P

1 k 1 1,- - P

diesterase sple~/

PJ P

k g a s e

2 HOJps 3-Mononwleotides

32p

4

OH

5'-Mononmleotides

FIG.6. Scheme for nearest neighbor analysis of phosphodiesters formed in the ligase reaction. From Weiss et al. (66a).

TABLE I11 NEARESTNEIGHBORANALYSISOF NUCLEOTIDES JOINED IN LIGASE REACTION"^^ DNA (Fig. 6) prior to the action of ligase 3'-Nucleotides 5'-Nucleotides 5'-Nucleotides DNA (Fig. 6) after the action of ligase

Nucleotide

(%I

(%I

(%)

dAMP d T MP dGMP dCMP

31

22 59

22

44 11

14

8 11

57 9 12

From W e i s et al. (664. A T? DNA preparation (300 mpmoles) containing 90% (the other sePwas terminal) of its a*Pin internal phosphomonoesters was incubated in the standard ligase reaction mixture for 30 min with 0.01 unit of DNA ligase. An aliquot of the reaction mixture was subjected to the standard assay procedure; 90% of 3 2 P in the DNA became insusceptible to phosphatase. The incubation mixture was dialyzed, incubated with phosphatase for 30 min a t 65" to remove any remaining phosphomonoesters. The protein was extracted with phenol, and the DNA was dialyzed against four changes of 0.01 M tris-HC1 buffer (pH 7.6)-0.05 M NaC1. One aliquot of DNA was hydrolyzed completely to 5'-mononucleotides (see Fig. 6) by the consecutive action of pancreatic DNase I and venom o-exonuclease. Another aliquot was hydrolyzed to 3'-mononucleotides by the action of micrococcal nuclease and spleen a-exonuclease. The only labeled nucleotides were those adjacent to the original DNase I cleavage (Fig. 4).

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which was originally inflicted by DNase I (Figs. 4 and 6). The data of Table I11 show that all four bases are present, but A and T are significantly favored. Little can be said concerning the location of nicks relative to the center of the molecule. Present evidence suggests th a t central locations are strongly favored (49, 5 3 ) . This is supported by inability to detect any sign of exonucleolytic character in the early phases of the DNase I reaction (67).A surprising and as-yet unexplained result of the latter work was the finding that in comparison to other nucleases DNase I showed an exceptionally high hyperchromic shift per cleavage during the early phase. As the reaction progressed a plateau was reached, even though titrimetry indicated a continuation of the reaction. One is tempted to speculate that the early nicks occur in such regions where the unwinding of an extended portion of the helix is possible (e.g., A,Trich regions). On the other hand, the cessation of optical changes prior to titrimetric changes suggests that a considerable portion of the oligonucleotides remains double stranded. An elegant study of the kinetics has been performed on single-stranded biosynthetic polymers (68),prepared by the method of Bollum et al. (69). Three polymers were used : (1) d ( [ 3H]PA) (PA)lzl (2) . d ( ['HI PA) (PA)llii ( [ 2-14C]PA) 4 ~ ;7 and (31 d ( [3HI PT) (PA) i ~ The digestion was carried out in the presence of Mg2+ and allowed to proceed in a pH stat until 10% of internucleotide bonds were hydrolyzed. The reaction was stopped by heating on a steam bath. The size of the products in the digestion mixture was determined by means of chromatography on a column of Bio-Gel P-60 previously calibrated with oligomers. The overall conclusion (58) is that the rate of attack on diester bonds within 10 nucleotides of an end is much smaller than on bonds in the central region, if the substrate molecule is several multiples of 10 nucleotides or less in length, but that this discrimination disappears as the substrate length increases. In connection with autoretardation it is interesting to note that with a single-stranded homopolymer, there is a greater probability of producing fragments larger than 10 than of producing shorter fragments. Fragments of about 15 are attacked slower than the larger ones. All these findings confirm autoretardation and extend it into a phase of the reaction, in which little or no double-stranded substrate remains. The observation that short fragments (less than 10) 57. E. J. Williams, S.-C. Sung, and M. Laskowski, Sr., JRC 236, 1130 (1961). 58. D. E. Hoard and W. Goad, J M B 31, 595 (1968). 59. F. J. Bollum, E. Goreniger, and M. Yoneda, Proc. Natl. Acad. Sci. U.S. 51, 853 (1963).

308

M. LASKOWSKI, SR.

are formed faster in the earlier than in the latter stages of the reaction implies that DNase I either recognizes structure up to 100 A long (3 x 10 oligomers) or recognizes such aspects of the tertiary structure of homopolymers that exist in the long, but not in short, fragments. As in the experiment of Bollum (11), Hoard and Goad ( 5 8 ) , observed that the thymidylic acid fragment was deiraded faster than the corresponding fragment containing adenylic acid. This was in contrast to the report of Ralph et al. (60). Since Bollum and Hoard and Goad used Mg2+whereas Ralph et a2. used Mn2+as activating cation, it seems possible that the observed differences were caused by the nature of the metal (see Section V ) . Entirely different kinetics characterize the terminal phase of the reaction. At this phase, the remaining substrates are quite resistant. Presumably, the end point of the reaction is primarily affected by the concentration of the activation ion. VII. Speciflcity

The previous characterization of specificity of DNase I (26, H )was arrived a t from the analysis of digestion products at the time of termination of the reaction. Three tacit assumptions were made, all of which are probably false: (1) the specificity does not change during the whole course of the reaction, (2) the nature of the activating ion does not influence specificity, and (3)the end point of the reaction is characteristic for the enzyme and is independent of the medium (ions, pH, type of substrate, etc.) Although the earlier data are valid for the conditions under which they were collected their generality needs to be reexamined. A long time ago, the work in the Sinsheimer’s laboratory (61-63)and in ours (64) established that among the products present a t the so-called termination of the reaction, dinucleotides of the type pPu-pPy were either rare or absent. This was interpreted as evidence that the Pu-pPy bond is the most susceptible to DNase I [for details, see reviews (10, 27, 30)1. This conclusion rests on the validity of the first assumption (see above). The recent work of Scheffler et al. (65) is the only work that supR. K. Ralph, R. A. Smith, and H. G. Khorana, Biochemistry 1, 131 (1962). R. L. Sinsheimer and J. F. Koerner, JACS 74, 283 (1952). R. L. Sinsheimer, JBC 208, 445 (1954). R. L. Sinsheimer, JBC 215, 579 (1955). M. Privat de Garilhe, L. Cunningham, U.-R. Laurila, and M. Laskowski, Sr., JBC 224, 751 (1957). 65. I. E. Scheffler, E. L. Elson, and R. L. Baldwin, J M B 36, 291 (1968). 60. 61. 62. 63. 64.

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ports this premise. The authors digested biosynthetic d (A-T) polymer with DNase I in the presence of Mg” and found only the products of the type d(pTpA),, where n was any integral number. No fragments starting with A, and no odd numbered fragments were detected. These findings show that only the cleavages between A and p?” were occurring throughout the whole course of the reaction, and no other cleavage. This agrees with the postulated specificity Pu-pPy. However, in many respects d (A-T) polymer behaves differently from a representative natural DNA with its four bases. The most obvious difference is its complementarity, which allows it to retain double-stranded conformation essentially throughout the reaction. To accomlish the double-strand scission the hydrolysis of an identical A-pT bond is required. The work on d(A-T), suggests that synthetic polymers may be most useful in elucidating some aspects of base specificity. The conclusion, however, that specificity remains unchanged throughout the whole course of the reaction is not directly transferable to DNA, where early cleavages appear to be considerably more specific than the later ones. I n Section VI the comparison of the rate of hydrolysis of (dA), and (dT), was discussed. The only way to account for the contradictory findings in different laboratories is to ascribe the differences to the activating ion used. The evidence exists that during the late phase of the reaction factors other than the Pu-pPy specificity determine the lability of the internucleotide bond. Thus, Potter et al. (42) easily digested d-ApApTp to d-ApA and d-pTp, whereas Khorana (43) observed that d-CpApT was resistant to DNase I. Both compounds had Pu-pPy sequence in the p - y positions, but differed with respect to 3’-terminal phosphate, which exerts a labilizing influence on the proximal internucleotide bond (see below). Doubts concerning the validity of the second and third premise stem from two sources: studies of the effects of metals on kinetics of DNase I, and from a comparison with other nucleases. Hurst and Becking (66-68) and Hacha and Fredericq (69) showed that whereas Mgz+ and Mn2+ both accelerated the action of DNase I, each led to a different mixture of products. Both groups suggested that DNase I may be a mixture of a DNase and an oligonucleotidase. I n view of Bollum’s findings (11) the probable explanation for these phenomena is that Mn2+ and Mg2+ affected susceptibility of different bonds in the substrate. The experiments in which a direct effect of concentration of a divalent 66. G. C. Becking and R. 0. Hurst, Can. J . Biochem. Physiol. 40, 166 (1962). 67. R. 0. Hurst and G. C. Becking, Can. J . Biochem. Physwl. 41, 469 (1963). 68. G. C. Becking and R. 0. Hurst, Can. J . Bwchem. P h y h l . 41, 1433 (1963). 69. R. Hacha and E. Fredericq, Bull. SOC. Chim. Belges 72, 580 (1963).

310

M. LASKOWSKI, SR.

cation on the end point of the reaction was demonstrated have been performed so far only with micrococcal nuclease (7). It seems likely, however, that with all DNases requiring a divalent cation, the requirement increases as the reaction proceeds. One issue was so far avoided, namely, the origin of mononucleotides. I n a digest stopped a t end point of the fast reaction the digestion mixture contains about 1% of mononucleotides. The maximum amount of mononucleotides ever observed after an exhaustive digestion was 5% ( 6 ) . All four mononucleotides are present but in different amounts. Ralph et al. (60) concluded that the smallest unit from which mononucleotides can be derived is a tetranucleotide, because in agreement with others (6) they found di- and trinucleotides resistant to large doses of DNase I . There is only indirect evidence suggesting that mononucleotides may originate from the o terminus (41). Summarizing the present status of our knowledge of the specificity of DNase I it is necessary to reemphasize the difference between the early and the terminal phase of the reaction. The only exception is biosynthetic d(A-T), (66). With DNA as substrate the early cleavages are directed toward the center of the molecule and are predominantly singlestrand nicks. By analogy to other nucleases one should expect that they are specific also with respect t o the adjacent bases. I n the latter part of the reaction the Pu-pPy bond is preferentially cleaved. The reaction can be carried to the stage when products are essentially a mixture of dinucleotides and trinucleotides. At this stage the term preferentially cleaved linkage is obviously nonapplicable.

VIII. Physiological Role

As yet no definite physiological role can be assigned to DNase I. The role of such an enzyme in digestion is testified by its presence in the pancreatic juice. However, the presence of DNase I activity in practically all other tissues casts doubt that the digestive function is its major role. The prevailing opinion assigns the major function of DNase I t o inflicting nicks during the early stages of hydrolytic attack on DNA. Thus participation in repair phenomena rather than complete digestion appears to be its major function. ACKNOWLEDOMENTS The experimental work referred to in this article and performed in our laboratory was generously supported by the American Cancer Society PRP-30 and E-157, by

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the U. S. Atomic Energy Commission AT(30-1)3630, and the National Science Foundation GB-6058. The author is indebted to Dn. Bollum, Moore, Richardson, Sinsheimer, and Weinfeld for the critical reading of the manuscript, and to Drs. Lindberg, Moore, and Richardson for permission to reproduce their data.