Restriction endonuclease cleaving palindromic DNA

The present invention teaches restriction endonuclease Asp 718 which cleaves DNA in the palindromic recognition sequence at the sites indicated by the arrow: ##STR1## The endonuclease, obtained from Achromobacter species 718, DSM 2969, is useful in obtaining DNA fragments, e.g., for determining nucleotide sequences and other types of analysis of DNA.

The present invention is concerned with a new Type II restriction 
endonuclease (Asp 718), with a process for obtaining it and with the use 
thereof. 
Type II restriction endonucleases are endodesoxyribonucleases which 
recognise certain DNA sequences and are able to split at definite 
positions. Certain phosphodiester bridges are thereby hydrolysed in the 
target sequence, namely, one in each polynucleotide strand. Type II 
restriction endonucleases are, therefore, valuable for the analysis of DNA 
molecules. 
Specific Type II restriction endonucleases are admittedly known for 
numerous DNA sequences but there is still a need for the provision of 
further Type II restriction endonucleases which cleave DNA sequences in 
those positions which hitherto have not been cleft by any of the known 
restriction endonucleases. 
Therefore, it is an object of the present invention to provide a new 
restriction endonuclease which is able specifically to recognise a 
sequence and is able to cleave at a new position. 
Thus, according to the present invention, there is provided a restriction 
endonuclease which is characterised by the palindromic recognition 
sequence 
##STR2## 
and by the cleavage position defined by the arrow. 
The cleavage position is such that 5'-overhanging individual strand ends 
result. This makes possible a radio-active marking of the resulting 
fragments on the 5' end with the enzyme T4 polynucleotide kinase and also 
on the 3' end with the help of the enzyme Klenow polymerase. This is, for 
example, advantageous in the case of sequencing experiments. 
The new Type II restriction endonuclease according to the present 
invention, hereinafter called Asp 718, has a temperature optimum of from 
35.degree. to 39.degree. C. and a pH optimum at 8.5.degree./37.degree. C. 
in tris/HCl buffer. Further optimum reaction parameters are 75 mmol/liter 
NaCl, 6 mmol/liter Mg.sup.2+ and 6 mmol/liter 2-mercaptoethanol. 
The presence of Mg.sup.2+ is necessary for the activity of the enzyme. 
As mentioned above, the enzyme acts upon palindromic sequences and thus 
recognises a self-complementary nucleic acid sequence in which the 
complementary strand of the double strand has the identical sequence in 
the counter direction. 
The recognition sequence and the point of cleavage can be ascertained as 
follows: 
The DNA of Virus SV40 (BRL) is linearised with Hpa II by cleavage at 
position 282. Both strands of this linearised DNA are terminally marked in 
two parallel, different reactions. The (-) strand is phosphorylated on the 
5' end at position 284 with gamma-[.sup.32 P]-ATP and T4 polynucleotide 
kinase. In the second reaction, the complementary (+) strand is lengthened 
by one nucleotide on the 3' end at position 282 with alpha-[.sup.32 
P]-dCTP and Klenow polymerase. Therefore, the marked (+) strand ends at 
position 283. Both differently marked DNAs are subsequently each split 
with Bgl I at position 5171. From each of the resultant 5'-and 
3'-terminally marked fragments 4892(5')/4889(3') and 351(5')/354(3'), 
(length of the fragments referred to the marked individual strands) is 
respectively isolated the 351 bp (5') and 354 bp (3') fragment (position 
5176 to 284(5'); position 5172 to 283(3')). The 5'-marked fragment is 
sequenced. 
Additionally, in each case an aliquot of the isolated 351 bp (5') or 354 bp 
(3') fragments is split with the enzyme according to the present invention 
and the length of the 5'- and 3'-marked individual strands determined in 
the sequence gel by comparison with the 5'-sequence director. On the 
5'-terminally marked (-) strand there is thereby given the cleavage 
position 234 and on the 3'-terminally marked (+) strand the cleavage 
position 230. 
The length determination of the 5'-marked (-) individual strand of the Asp 
718/Hpa II fragment is carried out in the following manner: 
The (-)-individual strand 5'-marked on position 284 runs identically with 
the inner and thus 3'-positioned "G" on position 234 of the 5'-sequence 
director within the recognition sequence 5'-GGTACC-3'. Therefore, the 
5'-marked individual strand terminates with the nucleotide G of the (-) 
strand at position 235 of the recognition sequence. The point of cleavage 
of Asp 718 on the 5'-marked (-) strand is thus between the nucleotide G at 
position 234 and G at position 235. 
The length of the complementary 3'-marked (+) individual strand of the Asp 
718/Hpa II fragment is determined analogously. The (+) individual strand 
3'-marked at position 283 runs identically with the inner and thus 
5'-positioned "C" at position 231 of the 5'-sequence director within the 
recognition sequence 5'-GGTACC-3'. The 3'-marked individual strand 
terminates, therefore, with the nucleotide G of the (+) strand at position 
231 of the recognition sequence. The point of cleavage of Asp 718 on the 
3'-marked (+) strand is thus between the nucleotide G at position 230 and 
G at position 231. 
According to the present invention, Asp 718 is obtained by culturing 
Achromobacter species DSM 2969 and the enzyme obtained from the cells. For 
obtaining it, there can be used the conventional biochemical purification 
methods, whereby, in the particular fractions obtained, the presence of 
the enzyme can easily be detected on the basis of the cleavage of its 
recognition sequence. As substrate, there can be used, for example, 
lambda-DNA. The DNA fragments obtained are separated electrophoretically 
in agarose gel in the buffer systems conventional for the fragment 
separation in the presence of ethidium bromide. 
The organism Achromobacter spec. DSM 2969 used for obtaining the enzyme 
grows aerobically in standard medium I, which is described hereinafter in 
detail in Example 1. 
The organism is gram negative. The cells are colloidal (0.5 to 2.0 .mu.m.) 
and are usually present individually. The temperature optimum is from 
25.degree. to 37.degree. C. The doubling time is about 1 hour. 
In a preferred embodiment of the process according to the present 
invention, the cells are digested, the extract is mixed with streptomycin 
sulphate until precipitation is complete, the precipitate is separated off 
and the supernatant is recovered. 
For the digestion, there can be used the conventional mechanical and 
chemical methods, for example high pressure dispersion, ultrasonics or 
enzymatic digestion. 
The high purification of the streptomycin supernatant containing the new 
enzyme preferably takes place by affinity chromatography, molecular sieve 
fractionation and via cation exchangers. As molecular sieve material, 
there has proved useful the product commercially available as Ultrogel ACA 
34, which is an acrylamide/agarose heteropolymer of 3% acrylamide and 4% 
agarose. 
As cation exchangers, there are preferably used phosphate group-containing 
substances, preferably carbohydrates, for example cellulose phosphate and 
the like. For the affinity chromatography, there has proved to be 
especially useful carrier-fixed heparin, for example heparin-sepharose CL 
6 B (Pharmacia).

The following Examples are given for the purpose of illustrating the 
present invention: 
EXAMPLE 1 
Achromobacter species DSM 2969 is allowed to grow aerobically at 30.degree. 
C. for 10 hours in standard medium I, which is described in detail 
hereinafter, and then harvested in the late logarithmic phase. 200 g. of 
the cell paste thus obtained (about 20 g. dry mass) are suspended in 500 
ml. of digestion buffer (40 mmol/liter Tris/HCl, pH 7.6.degree./4.degree. 
C.; 0,1 mmol/liter EDTA (ethylenediamine-tetraacetic acid); 7 mmol/liter 
2-mercaptoethanol and 0.2 mmol/liter PMSF (phenylmethanesulphonyl 
fluoride)). 
The cells are then digested twice by high pressure dispersion in a 
pre-cooled pressure cell at 1100 bar. 
The digestion suspension is mixed with 10% streptomycin sulphate solution 
until precipitation is complete. After leaving to stand for 30 minutes at 
4.degree. C., the precipitate formed is centrifuged off for 120 minutes at 
27300 g and discarded. 
The standard medium I has the following composition: 
______________________________________ 
distilled water 1000 ml. 
special peptone 15.6 g. 
yeast extract 2.8 g. 
sodium chloride 5.6 g. 
glucose 1.0 g. 
______________________________________ 
pH 7.4 to 7.6 
Fermentation conditions: 
150 liter Chemak fermenter 
100 liter working volume 
stirrer 450 r.p.m. 
air 0.08 Vvm 
temperature 30.degree. C. 
amount of inoculum 10% 
Yield: 60 g. of dry mass/100 liters of culture. 
EXAMPLE 2 
Streptomycin supernatant obtained according to Example 1 is chromatographed 
on an affinity chromatography column equilibrated with TEMG buffer 
(Heparinsepharose CL 6 B/5 cm..times.28 cm.). After washing with four 
column volumes of TEMG buffer, the enzyme is eluted with a linear TEMG 
gradient with 0 to 1 mol/liter of sodium chloride. The enzyme elutes in 
the fractions with 0.45 to 0.6 mol/liter sodium chloride. The active 
fractions are combined and precipitated out with solid ammonium sulphate 
up to a degree of saturation of 80% (w/v). The precipitate is left to 
stand for 70 hours at 4.degree. C. 
The precipitate thus obtained is centrifuged off for 60 minutes at 27,300 
g, taken up with TEMG buffer and applied to an Ultragel AcA-34 molecular 
sieve column with the dimensions 2.times.100 cm. This column is eluted 
with TEMG buffer+0.5 mol/liter sodium chloride and the eluate fractions 
with Asp 718 activity are combined. 
The combined fractions are dialysed against TEMG buffer and chromatographed 
on a cation exchanger column equilibrated with TEMG buffer (cellulose 
phosphate P11/3.times.10 cm.). After washing with two column volumes of 
TEMG buffer, the enzyme is eluted with a linear TEMG gradient of 0 to 1 
mol/liter sodium chloride. Asp 718 elutes between 0.4 and 0.6 mol/liter 
sodium chloride. 
The active fractions are combined and dialysed against 20 mmol/liter 
Tris/HCl buffer, pH 7.6.degree./4.degree. C.; 0.1 mmol/liter EDTA; 10 
mmol/liter 2-mercaptoethanol; 100 mmol/liter sodium chloride; 100 
.mu.g./ml. bovine serum albumin (BSA) and 50% glycerol and stored at 
-20.degree. C. 
Activity about 5 MU Asp 718 (activity definition: 1 U=1 .mu.g. 
lambda-DNA/hour at 37.degree. C. completely split). 
Activity determination 
Into a mixture of 5 .mu.l. incubation buffer, containing 0.03 mol/liter 
Tris/HCl buffer, pH 8.5.degree./37.degree. C.; 0.03 mol/liter magnesium 
chloride; 0.375 mol/liter sodium chloride; 0.03 mol/liter 
2-mercaptoethanol and 0.5 mg./ml. BSA are introduced 14 .mu.l. water and 5 
.mu.l. lambda-DNA (4 OD/ml.), as well as 1 .mu.l. Asp 718 solution (1 
U/.mu.l.; dilution with storage buffer). 
The solution is incubated for 1 hour at 37.degree. C., cooled on ice and 
mixed with 5 .mu.l. of a cold stop solution containing 7 mol/liter urea, 
20 wt./vol. % saccharose, 0.06 mol/l EDTA and 0.01 wt./vol. % bromophenol 
blue. 10 .mu.l. of this mixture are taken and mixed with 20 .mu.l. of 
diluted stop solution (above stop solution diluted 1:3 v/v with 0.02 
mol/liter EDTA). This solution is heated to 65.degree. C. for 20 minutes, 
stopped by pouring on to ice and 20 .mu.l. thereof separated 
electrophoretically on a 0.6% agarose gel in 16 hours at 50 V. The bands 
obtained are identified by comparison with appropriate DNA length 
standards.