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Itzhaki,R. F. (1971), Biochem. J . 122,583. Kleiman, L., and Huang, R. C . C. (1971), J . Mol. Biol. 55, 503. Kleiman, L., and Huang, R. C. C. (1972), J . Mol. B i d . 6 4 , l . Klein, F., and Szirmai, J. A. (1963), Biochim. Biophys. Acta 72,48. Klotz, I. M., and Hunston, D. L. (1971), Biochemistry IO, 3065. Le Pecq, J. B. (1972), Methods Biochem. Anal. 20,41. Le Pecq, J. B., and Paoletti, C. (1965), C. R . Acad. Sei. 260, 7033. LePecq, J. B., and Paoletti, C. (1967), J . Mol. Bid. 27,87. Le Pecq, J. B., and Paoletti, C. (1971), J . Mol. Biol. 59,43. Li, H. J., and Crothers, D. M. (1969), J . Mol. Biol. 39,461. Lurquin, P. F., and Seligy, V. L. (1972), Biochem. Biophys. Res. Commun. 46,1399. Minyat, E. E., Borisova, 0. F., Vol’kenshtein, M. V., and Georgiev, G. P. (1970), Mol. B i d . 4,234. Mirsky, A. E. (1971), Proc. Nut. Acad. Sci. U. S. 68,2945. Mirsky, A. E., and Silverman, B. (1972), Proc. Nar. Acad. Sci. U. S. 69,2115. Murray, K . (1969), J . Mol. Bid. 39, 125. Murray, K., Bradbury, E. M., Crane-Robinson, C . , Stephens,
R. M., Haydon, A. J., and Peacocke, A. R . (1970), Biochem. J . 120,859. Parker, C. A., and Rees, W. T. (1960), Analyst 85,587. Perrin, R. (1929), Ann. Phys. (Paris) 12,169. Rolfe, J., and Moore, S. E. (1970), Appl. Opr. 9,63. Scatchard, G . (1949), Ann. N . Y . Acad.Sci. 51,660. Schwimmer, S., and Bonner, J. (1965), Biochim. Biophys. Acta 108,67. Smart, J. E., and Bonner, J. (1971), J. Mol. B i d . 58,675. Spelsberg, T. C., and Hnilica, L. S. (1971), Biochim. Biophys. Acta228,212. Wahl, Ph., Paoletti, J., and LePecq, J. B. (1970), Proc. Nut. Acad. Sci. U . S. 65,417. Ware, W. R. (1971), in Creation and Detection of the Excited State, Lamola, A. A., Ed., Vol. 1, Part A, New York, N. Y . , Marcel Dekker, p 21 3. Waring, M. J. (1965), J. Mol. Biol. 13,269. Weber, G . (1952), Biochem.J. 51, 145. Weber, G . , and Teale, R. W. J. (1957), Trans. Faraday SOC. 53, 646. Williams, R . E., Lurquin, P. F., and Seligy, V. L. (1972), Eur. J . Biochem. 29,426.
Action of Micrococcal Nuclease on Chemically Modified Deoxyribonucleic Acid? N . W.
Y.Ho and P. T. Gilham*
ABSTRACT: The exposure of alkali-denatured DNA to the reagent N-cyclohexyl-N’- @ - (4 - methylmorpho1inium)ethylcarbodiimide (Cmc) p-toluenesulfonate results in the substantial blocking of the thymidine and deoxyguanosine moieties of the polynucleotide chains with Cmc groups. The hydrolysis of this modified DNA with micrococcal nuclease followed by the removal of the blocking groups produces a series of oligonucleotides, whereas, under the same enzymatic conditions, unmodified DNA is reduced to mono- and dinucleotides. The oligonucleotides in various size groups have been analyzed by degradation with alkaline phosphatase together with snake venom phosphodiesterase or spleen phosphodiesterase to determine their chain lengths and the identity of their 5’- and 3’-terminal nucleotides. In addition, the tetranucleotides, pdA-dT-dA-dT and pdT-dA-dT-dA, and their di-Cmc derivatives have been digested with the nuclease and the resulting products have been identified. From these
I
n comparison with the sequence analysis of RNA molecules the direct determination of the primary structure of DNA chains has presented a somewhat more difficult problem. While a number of sequences of some small sections of D N A molecules have already been assigned it has become apparent that one of the main difficulties in this work arises from the f From the Biochemistry Division, Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907. Receiued August 9, 1973. Supported by Grant GM18533 from theNationalInstitutesofHealth.
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results a specific pattern for the action of the nuclease on the modified DNA can be derived: (i) a preferential endonucleolytic cleavage takes place at the -Np-dA- bonds (where N is a modified or unmodified deoxyribonucleoside); (ii) each oligonucleotide, so formed, can then undergo the normal exonucleolytic degradation at its 3 ’ terminal depending on whether it contains a modified or unmodified nucleotide at its 3’ terminal; (iii) those oligonucleotides that contain a blocked dT or dG at their 3’ terminals tend to be resistant to this exonucleolytic action while the rest of the oligonucleotide species are subject to the stepwise removal of unblocked nucleotides until a blocked dT or dG is located at their 3 ’ terminals. This restricted activity of micrococcal nuclease constitutes a method for the specific cleavage of polydeoxyribonucleotides that is expected to be of some value in future studies on the sequence analysis of DNA.
relative lack of enzymatic methods for the cleavage of polydeoxyribonucleotides into specific smaller fragments for subsequent analysis. In the case of the work on RNA, the relative ease with which sequence information can be obtained depends, in large part, on the availability of base-specific endonucleases such as the ribonucleases A, TI, and U‘L.In addition, the reversible chemical modification of certain nucleotides within an RNA chain can be used to induce even greater specificity on the action of these ribonucleases. There are no known counterparts of these ribonucleases that
ACTION OF NUCLEASE O N MODIFIED
DNA
could be used for the specific cleavage of D N A molecules, and the present work was begun with the expectation that prior chemical modification of DNA might result in the induction of base specificity in the cleavage mechanisms of some of the common deoxyribonucleases. Uridine, thymidine, guanosine, and deoxyguanosine and their S'-phosphates react readily with the cation of the reagent N-cyclohexyl-N~-~-(4-methylmorpholinium)ethylcarbodiimide p-toluenesulfonate (Cmc p-toluenesulfonate),' at pH 8-9,and the resulting derivatives can be converted back to the corresponding nucleosides and nucleotides by exposure to dilute ammonia (Gilham, 1962; Ho and Gilham, 1967). The conditions necessary for the chemical modification of these nucleoside moieties within a polynucleotide strand, as well as the conditions required for the subsequent removal of FIGURE 1: Elution patterns bom the chromatography of micrococcal the blocking groups, are of a sufficiently mild nature that the nuclease digest on a column (50 X 0.8 cm) of DEAE-Sephadex. structural integrity of the polynucleotide chain can be preElution was effected with 1000 ml of 0.005 M Tris-C1-7 M urea(pH 8) containing a linear gradient of 0-1.0 M sodium chloride at a flow served. Thus, it has been possible to use this type of chemical rate of 10 ml/hr: (A) Cmc-DNA digested with 100 units of micromodification to restrict, in a specific way, the normal nucleococcal nuclease at 37" for 1 hr under the conditions described in lytic activity of ribonucleases A, TI, and UP toward the Experimental Section; (B) heat-denatured DNA digested under RNA (Lee et al., 1965; Naylor et al., 1965; H o and the same conditions. Gilham, 1967; H o et al., 1969), and these techniques have been used in studies on the nucleotide sequences in a number modified nucleic acids to the action of the various nucleases it of RNA molecules. Similarly, denatured DNA has been is not possible to obtain accurate values for the percentages of derivatized with the carbodiimide reagent (Augusti-Tocco and thymidine and deoxyguanosine moieties that have been modiBrown, 1965), and Drevitch et al. (1966)have observed that fied by the reagent. Rough estimates of the degrees of modifisuch derivatization of DNA decreases the number of bonds cation can be achieved by degrading the Cmc-DNA with a that would normally be subject to cleavage by pancreatic mixture of the enzymes, deoxyribonuclease I, micrococcal deoxyribonuclease. nuclease, snake venom phosphodiesterase, spleen phosphoMicrococcal nuclease, a phosphodiesterase that is capable of diesterase, and alkaline phosphatase. The combined action of hydrolyzing either DNA or RNA to nucleoside 3 '-phosphates, these enzymes would be expected to convert all the unmodihas been extensively studied. The results of the work on its fied nucleotide moieties in the DNA to nucleosides with the cleavage specificity have been reviewed by Anfinsen et al. possible exception of some of the nucleotides that are con(1971)and, on the basis of evidence from a number of laboranected to the 5' positions of modified nucleotides within the tories, they have concluded that, in native DNA, the -Np-dTpolynucleotide chain (Naylor et al., 1965). The mixed enzyme and -Np-dA- bonds are preferentially attacked while, in hydrolysis of Cmc-DNA prepared at room temperature redenatured DNA, the order of cleavage appears to be nearly sulted in the conversion of about 7 0 x of the material to random. However, a recent study in this laboratory involving nucleosides and modified nucleosides with the remainder of a detailed analysis of the terminal nucleosides appearing i n the products appearing as highly modified oligonucleotides. polynucleotide fragments during digestion with this nuclease An analysis of these products indicated that more than 70% confirms that the preferential endonucleolytic cleavage of of the deoxyguanosine and more than 80 % of the thymidine -Np-dT- and -Np-dA- bonds occurs also with the denatured moieties in the DNA had been modified by the reagent. form of DNA (Roberts et al., 1962). In addition, the enzyme is Cmc-DNA was treated with micrococcal nuclease and the known to possess exonucleolytic activity in that, subsequent blocking groups were subsequently removed with alkali. to its endonucleolytic action; the nuclease is capable of removHeat-denatured DNA was also treated with the enzyme under ing nucleoside 3 '-phosphate moieties from the newly formed the same conditions and the two sets of products were then 3' terminals of the polynucleotide fragments until the entire separated according to chain length by ion-exchange chronucleic acid is reduced to a mixture of mono- and dinucleomatography with solvents containing 7 M urea (Tomlinson and tides (Reddi, 1960; Sulkowski and Laskowski, 1962). Thus, Tener, 1963). The elution patterns (Figure 1) indicate that, in the absence of extra cleavage specificity that might be incompared with heat-denatured DNA, the Cmc-DNA conduced by chemical modification of the substrate, it would tains far fewer phosphodiester bonds that are susceptible to seem that the enzyme could have very little use in the sequence the nuclease action. The products from each digestion were analysis of DNA. combined into fractions as indicated in the elution patterns. Salmon sperm DNA was used for the present studies and a These fractions were then rendered free of salt and urea and number of experimental methods for its derivatization with the the oligonucleotide components of each fraction were analyzed Cmc reagent were investigated. The preferred method of with respect to their base compositions, average chain lengths, derivatization involves the alkaline denaturation of the DNA and their 3'- and 5'-terminal nucleosides. The method of followed by exposure of the product to a high concentration analysis (Ho and Gilham, 1973) consists of (i) the treatment of the reagent at room temperature for 20 hr. A more rapid of the fraction with alkaline phosphatase, (ii) the destruction of derivatization results from a similar treatment of the DNA at the enzyme in situ by exposure to alkali, (iii) the digestion of higher temperatures but it is accompanied by some cleavage the products with snake venom phosphodiesterase or spleen of the polynucleotide chains. Due to the resistance of Cmcphosphodiesterase, and (iv) the separation of the resulting nucleosides and nucleotides by ion-exchange chromatography. From the results corresponding to elution pattern A (Table I) 1 Abbreviation used is: Cmc, N-cyclohexyl-N'-~-(4-methylmorpho1inium)ethylcarbodiimide. it can be concluded that most of the larger oligonucleotides B I O C H E M I S T R Y ,V O L .
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TABLE I : Analysis of Terminal Nucleosides of
Elution Pattern
Fraction
A
I
A
I1
A A A B B B
111 IV V I
5 '-Terminal Nucleoside
dC
dA
dT
dG
dC
dA
dT
dG
1 .o 2.0 3.0 4.5 8.4 1. o 2.0 3.0
32 11 18 18 20 21 3 6
53 78 75 80 78 34 49 41
8 10 6
