The Journal of Biochemistry, Vol. 64 , No. 3, 1968

The two , Kl and K2, Isolated from Mycelia of Aspergillus oryzae*

II. Determination of 5•Œ and 3•Œ-Termini of the DNA Fragments Produced by DNases K1 and K2

By MIKIO KATO,** TADAHIKO ANDO and YONOSUKE IKEDA

(From the Microbiology Laboratory, Institute of Physical and Chemical Research, Yamato-machi, Saitama)

(Received for publication, February 5, 1968)

The DNA fragments produced from calf thymus DNA by treatment with DNases K1 and K2 were studied.

1. The Ki digest gave deoxyguanosine and deoxyadenosine when

treated with venom [EC 3.1.4. 1] and deoxyguanosine when treated with alkaline [EC 3.1.3. 11 and spleen phos

phodiesterase [EC 3.1.4.1]. It was presumed that DNase KI might preferentially split the phosphodiester-bonds between dG and dG and between dG and dA at the 5•Œside giving as the products oligo and

poly-nucleotides with 3•Œ terminal phosphate. 2. The K2 digest was studied by similar methods. DNase K2

seems to split preferentially the above mentioned bonds at the 3•Œside

giving as the products oligo and poly-nucleotides with 5•Œterminal phos

phate. 3. The K1 and K2 digests were fractionated on DEAE-cellulose columns. The digests consisted of single and double stranded DNA fragments with various molecular size. The large DNA fragments were comparatively poor in G.

4. When a double stranded DNA fraction from the K2 digest was

digested with an which specifically splits single stranded portion, there appeared a large percentage of 5•Œ deoxyguanylic acid. The non

digestible portion showed hyperchromism. It was presumed that the DNA fraction might consist of a double stranded portion and a single

stranded portion.

As reported in the previous paper (1), the This paper reports the structure of 5•Œand DNases K 1 and K 2 isolated from mycelia of 3•Œ-termini of the fragments. The termini were Aspergillus oryzae IAM 2645 split the nucleotide determined by digestion with snake venom chains of DNA to fragments of considerably phosphodiesterase [EC 3.1. 4. 1], alkaline phos

large molecule. A portion of the fragments phatase [EC 3.1.3.1], and spleen phosphodi shows hyperchromism. [EC 3.1.4.1] (2). The products were fractionated on a DEAE-cellulose column, and * This work was supported by a Grant in Aid their average chain length was estimated. from the Science and Technology Agency and a Grant MATERIALS AND METHODS from the Kyowa Fermentation Industry Co., Ltd. ** Present address : Kyowa Fermentation Industry Substrate-Calf thymus DNA (type I) purchased Co., Ltd., Oote-machi, Tokyo. from Sigma Chem. Co., was used. 329 330 M. KATO, T. ANno and Y. IKEDA

Enzymes-DNases KI and K2 purified 500 and examined by paper chromatography. 300 folds, respectively, were used. The methods used Determination of Average Chain Length-The length

for the preparation have been described previously (1). (number of nucleotides) was estimated from the ratio Snake venom phosphodiesterase and E. coli alkaline of terminal phosphate to total phosphate. The terminal

phosphatase were purchased from Sigma Chem. Co. phosphate was determined by NAKAMURA•ŒS method (4) Spleen phosphodiesterase was purchased from Wor after treatment with . The re thington Chemical Co. S1 was prepared from action mixture consisted of I ml of Kl or K2 digest

Takadiastase by the method of ANDO(3). (0.2 mg as DNA), 0.2 ml glycine buffer (pH 10.3), and KI and K2 Digests-KI digest: A mixture consist 0.03 ml of alkaline phosphatase (0.5 units/ml), and in ing of lOml of 0.2% calf thymus DNA , 4m1 of Tris cubated at 37•Ž for 2 hr. In the case where total HCl buffer (pH 9.0), 2 ml of 0.1 M MgSO4, and 4 ml phosphate was to be measured, the KI or K2 digest of DNase Ki (32 units/ml) was incubated at 37•Ž for was decomposed by the wet oxidation method (5). 60min. The viscosity was reduced to the lowest level Fractionation of 0ligonucleotides-A DEAE-cellulose by this treatment, and about 6% of the original DNA column (1.8x30cm) was washed with 2liters of 0.01 M turned to be soluble in 3m TCA (1) . To stop the ammonium-bicarbonate plus 0.01 M Tris-HCl (pH 8.6) reaction, the tube was chilled in an ice-water bath . solution and then loaded with 10 mg of digest (as DNA) K2 digest : Calf thymus DNA was digested with dissolved in 50ml of ammonium bicarbonate plus Tris DNase K2 (30 units/ml) under the conditions stated buffer solution, The charged column was washed with above using a buffer of pH 8.0. About 8% of the 100ml of the same buffer. Elution was done stepwise products was TCA soluble. with 150 ml each of 0.2 M, 0.4 M and 0,9 Mammonium Paper Chromatography-The system used for the study bicarbonate, 0.9m NaCl, and 0.5m KOH solution. of nucleosides consisted of n-butanol : ammonical aquor Five ml fractions were collected in small tubes. (0.6N)=6: 1, and that for nucleosides and nucleotides Determination of Base Composition-The samples (each consisted of isobutylic acid : ammonical aquor (0 .5 N)= about 0.2 mg as DNA) were dried in vacua and then 10: 6. Samples on a Whatman No . i filter paper were hydrolyzed with formic acid (98%, Merck Co.) at 175•Ž developed one-directionally at 25•Ž. The paper were for 30 min according to the prescription of WYATT (6). exposed to ultraviolet light, and nucleoside or nucleo The products were chromatographed by the one-dimen tide in each spot was extracted with I N HCI to be sional descending technique using a solvent mixture used for spectroscopical analysis, consisting of 2•Œ-propanol(65%, v/v) : HCI(36%) : water= Determination of 5•Œand 3•ŒTermini-The 5•Œand 3•Œ 70:20: 10. The bases produced were extracted with termini of the KI and K2 digests were determined by 0.1 a HCl and their amounts were measured spectro the methods described by COHN and VOLKIN (2) with photometrically. a slight modification.

5•Œ-Terminus-One mg of KI digest (as DNA) was RESULTS mixed with 0.1 ml of Tris-buffer (pH9 .0, 0.2m) and Terminal Nucleosides of the K 1 and K 2 Di 0.03 ml of snake venom phosphodiesterase (0 .5 units/ml). After incubation at 37•Ž for 3 hr gests-Fig. 1 shows the results of experiments, , the reaction was stopped by chilling. A 0.2 ml aliquot was concentrated in which the K 1 digest was treated with and spotted on a Whatman filter paper. The nucleo venom or spleen phosphodiesterase. As may side liberated from the 5•Œ-terminus was identified on be seen from the figure, the treatment with the paper by comparison with authentic samples . The venom phosphodiesterase gives deoxyguanosine 5•Œ-terminus of the K2 digest was determined by the and deoxyadenosine in the ratio of about 7:3. above stated method after pretreatment with alkaline Occasionally, a spot for deoxythymidine was phosphatase ; that is, a mixture consisting of 1 mg K2 found, but its amount was quite small (about digest (as DNA), and 0.01 ml of alkaline phosphatase one tenth of that of deoxyguanosine). On the solution (0.5 unit/ml) was incubated at 37•Ž for 30 min . other hand, the digest gave only deoxyguano The reaction was stopped by heating the mixture in sine when treated with alkaline phosphatase an autoclave at 105•Ž for 10min. 3•Œ-Terminus-The Kl or K2 digest was pretreated and spleen phosphodiesterase. The results with alkaline phosphatase as stated above suggested that the 5•Œ-end of the K 1 digest , and then might be terminated with either deoxyguano the solution was adjusted to pH6 .5 by addition of 0.2m acetate buffer (pH 4.0). The resultant mixture sine or deoxyadenosine and the 3•Œ-end with was incubated with 0.03 ml of Tris-HCl buffer (pH 7 .5, deoxyguanosine. The 3•Œ-end of K I digest was 0.2 M) and 0.03 ml of spleen phosphodiesterase (1 .0 units/ found to be phosphorylated . ml) at 37•Ž for 5 hr. The nucleosides liberated were In Fig. 2 are presented the results of Specificites of DNases 331 experiment, in which the K 2 digest was 1-2, 3-4, and about 7 nucleotides, respec treated with venom or spleen phosphodieste tively. The data are shown in Table 1. The rase. The treatment with alkaline phospha table also shows the base ratio in each frac tase and venom phosphodiesterase gave both tion. The G-content in the early fractions deoxyguanosine and deoxyadenosine, whereas the treatment with alkaline phosphatase and spleen phosphodiesterase gave deoxyguanosine only. Neither deoxyguanosine nor deoxy adenosine was found unless the digest had been pretreated with alkaline phosphatase.

The results suggested that the 5•Œ-end of the K 2 digest might be terminated with either deoxyguanosine or deoxyadenosine and the

3•Œ-end with deoxyguanosine. The K 2 digest was found to be phosphorylated at the 5•Œ-end. Fractionation of K I and K 2 Digests and Estimation of Average Chain Length of Each Frac tion-Fig. 3 shows the elution profiles from DEAE-cellulose columns. The yield of each fraction is indicated in the figure. The chain length of the oligonucleotides contained in 0.2m, 0.4 as, and 0.9 .t ammonium bicarbonate fractions was estimated from the ratio of terminal phosphate to total phosphate to he

Fin. 2. Paper chromatogram of nucleosides isolated from K2 digest after treatment with snake venom or splenic PDase. A : The K2 digest was treated with venom PDase after pretreatment with E. coli PMase. B : The K2 digest was treated with splenic PDase after pretreatment with E. coli PMase. C : The K2 digest was treated with venom PDase.

FIG. 1. Paper chromatogram of nucleosides isolated from K1 digest after treatment with snake Fin. 3. Elution pattern of digestion products on DEAE-cellulose column. venom or splenic PDase. A : The digest was treated with venom PDase. A : Column chromatography of DNase K1 digest. B : The digest was treated with splenic PDase. B : Column chromatography of DNase K2 digest. 332 M. KATO , T. ANDO and Y. IKEDA

TABLE I Average nucleotide number and base composition of the DNA fragments obtained from KI and K2 digests.

was considerably high and that in the last One ml of the 0.9 M NaCl fraction (about fraction was low. This specific pattern is 4 mg as DNA) from the K 2 digest was mixed, consistent with the expectation; if K 1 and in a dialyzation bag, with 0.5 ml of nuclease K 2 are splitting nucleotide chains of S 1 (150 units/ml) and 0.5 ml of 0.02 M acetate DNA preferentially at the G-G and G-A bonds , the early fraction should be G-rich. In the experiment shown in Fig. 4, the 0.9m NaCl fraction was heated under the condition described in the previous paper (1) . The fraction showed clear hyperchromism, suggestingthat the fragments contained in this fraction might be double stranded. The 0.5 M KOH fraction also showed hyperchro mism, but the ammonium bicarbonate frac tions did not.

Digestion of 0.9A, NaCl Fraction with Nu clease S 1-As stated above, the DNA fragment

contained in the 0.9 M NaCl fraction displayed hyperchromism. If the DNA fragments possess

single stranded portions on their both ends,

digestion of the DNA with an enzyme which specificically split single stranded DNA may Fio. 4. Hyperchromism of DNA-fragments contained in 0.9m NaGI fractions of K1 and K2 give a certain amount of mono-nucleotides. Moreover, if the DNA is terminated domi digests. -•›- Intact DNA nantly with G as speculated previously, the . -•£- 0.9m NaCl fraction of Kl digest digestion products may contain a large per . -•œ- 0.9 M NaCl fraction of K2 digest . centage of deoxyguanylic acid . Nuclease S 1 -- •£-- Renaturation curve of 0.9 M NaCl fracti used in the following experiment was an on of Kl digest. enzyme which specifically hydrolyzes single -- •œ-- Renaturation curve of 0.9M NaCl fraction stranded DNA to 5•Œmono-nucleotides (2) . of K2 digest. Specificites of DNases 333

of uncertainty whether the 0.9 M NaCl fraction is a mixture of double-stranded DNA frag ments and single-stranded DNA fragments or a mixture of double-stranded DNA fragments with single-stranded DNA portions. However, the fact that the fraction shows a relative absorbance index of 1.28 seems to support the latter alternative. Assuming that the fraction consists mainly of double-stranded DNA molecules with single-stranded portions, the ratio of single-stranded portions to double stranded portions is calculated to be 1 :4 (see the last experiment in the preceding section). As the average number of nucleotide pairs in the fragments is about 15 (Table I), the average length of single-stranded portions may be smaller than 3 nucleotides.

The K 1 digest gives rise to deoxyguano

sine and deoxyadenosine when treated with

alkaline phosphatase and spleen phosphodi esterase. Moreover, the digest is phosphoryl

ated at the 3•Œend. Hence, the DNase K 1 FIG. 5. Paper chromatogram of the nucleo seems to split preferentially the phosphodiester tides produced from the 0.9m NaCl fraction of K2 digest by further digestion with . bonds between dG and dG and between dG S: Sl treated sample. and dA at the 5•Œ side. DNase K 2 also attacks the same bonds,

but the enzyme is thought to split the phos buffer (pH 4.5, supplemented with 0.1 mm ZnC12), and the mixture was dialyzed against phodiester bonds at the 3•Œ-end. When DNA which had been digested with 50 ml of the same buffer at 37•Ž. Every one K 1 or K 2 extensively (120 min) was treated hour during the dialysis, the outer-solution was renewed. The fifth solution contained a with venom phosphodiesterase, a spot for de negligible amount of UV absorbing substance. oxythymidine was found on the paper besides those for deoxyguanosine and deoxyadenosine. The total amount of mono-nucleotides dialyzed It may be concluded, therefore, that the two was about 20% of the original DNA. DNases are which preferentially The composition of the nucleotides in the attack the dG-p-dG and dG-p-dA bonds. one-hour digest was studied chromatography The preference seems to be in the order of cally using isobutylic acid: ammonical aquor dG-p-dG, dG-p-dA, dG-p-dT. system. As seen in Fig. 5, the spot corres Our enzymes have been compared with ponding to 5•Œdeoxyguanylic acid was most apparent. the following enzymes reported in the litera ture. But, no similarity has been found. DISCUSSION Venom nuclease (7), Micrococcous pyogenes nu The K 1 and K 2 digests reported in the clease (8-22), mung bean nuclease (23), previous paper (1) are mixtures of oligo and mammary tumors DNase (24 ), calf spleen poly-nucleotides. The DNA fragments showing DNase (25), crab testes DNase 26), octopus hyperchromic effect have been separated from nuclease (27), E. coli DNase (28), Neurospora others by column chromatography. The result crassa DNase (29), Streptococcous DNase of digestion with nuclease S 1 suggests that (30-39), phage T2 DNase (40), Azotobacter the fraction may consist of double-stranded agilis DNase (41, 42), Micrococcous lysodeikticus DNase (43), E. coli DNase (by infection of part and single-stranded part. It is a matter 334 M. KATO, T. ANDO and Y. 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