A restriction endonuclease from Haemophilus parainfluenzae degrades {varphi}X174 replicative form DNA into eight specific fragments, ranging from 1,700 to 150 base pairs and terminated specifically by deoxycytidylic acid

Johnson, Paul H.

Lee, Amy Shiu

Sinsheimer, Robert L.

English

Caltech Authors - Main

JOURNAL OF VROLOGY, Apr. 1973, p. 59689
Copyright @ 1973 American Society for Microbiology
Vol. 11, No. 4
Printed in U.S.A.
Production of Specific Fragments of 4X174Replicative
Form DNA by a Restriction Enzyme from
Haemophilus parainfluenzae,
Endonuclease HP
PAUL H. JOHNSON, AMY SHIU LEE, AND ROBERT L. SINSHEIMER
Division of Biology, California Institute of Technology, Pasadena, California 91109
Received for publication 6 November 1972
A restriction endonuclease from Haemophilus parainfluenzae degrades *X174
replicative form DNA into eight specific fragments, ranging from 1,700 to 150 base
pairs and terminated specifically by deoxycytidylic acid.
Several bacterial restriction deoxyriboendonu-
cleases have been described recently which pro-
duce unique fragments from high-molecular-
weight foreign DNA, are not associated with a
known modification activity, and require no
special cofactors other than divalent metal ions
(3, 6, 8). These enzymes will be very useful for
studying DNA structure and function. We are
interested specifically in the use of sequence-
specific deoxyriboendonucleases for the isolation
and nucleotide sequence determination of the
initiation sites for DNA replication and transcrip-
tion, and for the analysis of 4X174 replicating
intermediates.
Endonuclease HP was isolated from cultures of
Haemophilus parainfluenzae (strain originally
from Sol Goodgal and G. Leidy) by using a
modification of -the procedure developed by
Smith and Wilcox (8) for the purification of
endonuclease R from H. influenzae. The enzyme
eluted from a Biogel column (2.5 by 48 cm) be-
tween volumes of 180 and 230 ml. Fractions
containing endonuclease activity and A230/
Ano > 3 were pooled. After the ammonium
sulfate precipitation (8), the enzyme preparation
was desalted by chromatography on Sephadex
G-25. The enzyme was then eluted from a
phosphocellulose column at 0.2 M KCl.
Contaminating exonuclease activity present
at this stage of the purification was removed
by chromatography on DEAE-cellulose (What-
man DE52). For this purpose, a column was
equilibrated with 0.01 M potassium phosphate
buffer, pH 7.4, and the enzyme was applied
at 10 mg of protein per ml of column bed volume.
Elution was stepwise with three column volumes
each of 0.05 M, 0.1 M, and 0.2 M NaCl in 0.01 M
potassium phosphate, pH 7.4. Endonuclease HP
eluted at 0.05 M NaCl, whereas the exonuclease
activity eluted after 0.1 M NaCl. The DEAE
fractions were stored at 4 C until used. Endo-
nuclease R was purified as described by Smith
and Wilcox (8) and did not include DEAE-
cellulose chromatography.
'H-OX174am3 replicative form (RFI) DNA
was isolated from chloramphenicol-treated cells
and purified by Biogel chromatography, equi-
librium centrifugation in CsCl-ethidium bromide
density gradients, and sedimentation in isokinetic
neutral sucrose gradients. Details of the isolation
procedure will be described elsewhere.
Electrophoresis of DNA fragments was per-
formed in 5% polyacrylamide gels (1 by 20 cm)
formed in Plexiglas tubes. The buffer and
methods used were those described by Loening
(5) plus 0.1% (wt/vol) SDS. Electrophoresis was
performed at room temperature at a constant
potential of 45 V; running time was 1 h per cm of
gel length. To quantitate the radioactivity, gels
were frozen and cut into 1-mm segments by using
the Mickle gel slicer (Brinkman Instruments).
Gel slices were solubilized with toluene-Liquifluor
containing 10% NCS (Amersham-Searle), and the
radioactivity was measured in a Beckman liquid
scintillation counter.
End-group determination was performed as
described in the legend to Table 2. Polynucleo-
tide kinase was purified from T4 phage-infected
cells by using Richardson's original procedure (7)
modified by employing DNA-cellulose and Biogel
exclusion chromatography. Details will be pub-
lished elewhere.
Figure 1 shows the fractionation of an endo-
nuclease HP limit digest of jX174 RF DNA by
5% polyacrylamide gel electrophoresis. Improved
separation of fragments P1 and P2 can be ob-
tained by using gels of lower acrylamide concen-
596
VOL. 11, 1973
0
4
E
Mobility -_
FIG. 1. Polyacrylamide gel electrophoresis of
*OX174 RF DNA fragments produced by endo-
nuclease HP. The reaction mixture consisted of
0.1 ug of 'H-RFI, 7mM Tris, 7 mM mercaptoethanol,
50 mM MgCl2, pH 7.4; total volume, 100 pliters. A
O-pliter amount of endonuclease HP was added at
zero time and again at h. Digestion was at 37 C
for 10 h. To stop the reaction, EDTA ws added to
0.01 M. Sucrose and sodium dodecyl sulfate were
added to5 and0.5% final concentrations, respectively.
The mixture was incubated at 37 C for 10 min and
layered on a 6% polyacrylamide gel column (1
by 20 cm). Electrophoresis was as described in the
text. Mobility is the distance of migration in milli-
meters at the end of the run.
tration. The positions and proportioins of the
peaks do not change with increasing incubation
time or increasing enzyme concentration, indi-
cating that a limit digest of the DNA has been
produced.
Figure 2 shows that there is a linear relation-
ship between the logarithm of the integrated
counts in each peak (Fig. 1) and the electro-
phoretic mobility. Fragment P4 cointains twice
the mass expected, suggesting the presence of two
distinct fragments of similar size. A partial
separation of these two fragments can be ob-
tained with longer electrophoresis time.
Table 1 shows the size of the endonuclease
HP fragments calculated from the fraction of
total counts present in each peak. It has been
demonstrated that the molecular weights of
double-stranded DNA fragments determined
by mobility during polyacrylamide gel electro-
phoresis are in good agreement with values deter-
40
30
20 _
xb- 10
0Q-
0-
E 5
4
3
2
20 40 60 80 100
Mobility-._
FIG. 2. Relationship between mass and electro-
phoretic mobility for OX174 RF DNA fragments
produced by cleavage with endonuclease HP. The
mass is assumed to be proportional to the total num-
ber of counts in each peak and is plotted against
the distance migrated during the experiment (mo-
bility). The parallel line (---) is that calculated
for a peak with twice the mass; the point on this line
represents P4.
T.iBLE 1. Size of OX174 RF DNA fragments pro-
duced by cleavage with endonuclease HP
Fragment no.a Nucleotide pairsb
P1.......................... 1,670
P2.......................... 1,400
P3.......................... 825
P4.1, 4.2.......................... 470
P5.......................... 330
P6...................... 180
P7 ...: ............................ 150
a Fragment numbers correspond to those indi-
cated in Fig. 1.
b The niumber of nucleotide pairs is calculated
by assuming that the total is equal to 5,500 and
that mass is proportional to the total number of
counts in each peak.
mined by electronl microscopy and sedimenta-
tion velocity analysis (1, 2). The size and number
of fragments produced by endonuclease HP are
distinct from those produced by the restriction
endonuclease from H. influenzae, endonuclease
R. We have found that the latter enzyme pro-
duces at least 12 specific fragments from 6X174
*\ "\\0=
m=I
NOTES 597
J. VIROL.
TABLE 2. A comparison of the 5'-terminal nucleo-
tides of OX174 RF DNA fragments produced by
endonuclease HP and endonuclease Ra
Mol % nucleotide
Substrate Restriction at 5' endsenzyme
A T G C
*X174 RFI Endo HP <1 <1 <1 97
#X174 RFI Endo R 55 6 35 4
T7b Endo It 60 0 40 0
Lambda 44 7 45 4
a OX RF DNA (>98% form I) was degraded by
endonuclease HP in a reaction mixture consist-
ing of 7mM Tris (pH 7.5), 7 mM mercaptoethanol,
50 mM MgCl2, 50 mM NaCl, 12 ug of DNA, 125
uliters enzyme (0.1-0.5 units); total volume, 150
Aiters. Incubation was at 37 C for 14 h. The reac-
tion with endonuclease R was the same except
the MgCl2 concentration was 7 mM. The reaction
was stopped by incubation at 0 C with twofold
excess EDTA. The reaction mixture was adjusted
to 0.1 M Tris (pH 8.0) and 1 unit of alkaline phos-
phatase (Worthington; purified free from endo-
nuclease activity) per ml in a volume of 200
Aiters, and incubated at 37 C for 45 min. The
phosphatase was inactivated by addition of
ethylene-glycoltetraacetic acid to 6.5 mM fol-
lowed by incubation for 7 min at 65 C (Theodore
Live, personal communication). The reaction
volume was adjusted to 300 gliters and 10 mM
MgCl2, 20 mM mercaptoethanol, 1.7 mM potas-
sium phosphate, 70 mM Tris (pH 8.0), 15 nmol
of -yAT32P per ml (20 mCi/pmol, ICN), and 80
units of polynucleotide kinase per ml. The reac-
tion mixture was incubated at 37 C for 1 h and
stopped by a final concentration of 50 mM EDTA
and pyrophosphate. The labeled DNA was sepa-
rated from the unreacted ATP by exclusion
chromatography on a porous, glass bead column
(1 by 38 cm; Sigma G-240-250, approximate ex-
clusion limit 100,000) and precipitated at -20 C
for 12 h by two volumes of isopropanol and 0.3 M
sodium acetate, pH 5.5. The precipitate was col-
lected by centrifugation and dissolved in 100
gliters of 25 mM Tris (pH 7.5), 10 mM MgCl2, and
200 ug of pancreatic DNase I (Worthington WDPC
grade) per ml. Incubation was at 37 C for 30 min.
A 10-uliter amount of 1 M glycine (pH 9.5) and
20 pg of venom phosphodiesterase (Worthington,
purified by the procedure of Sulkowski and Las-
kowski [9]) were added, and incubation was con-
tinued for 30 min. The reaction was stopped by
50 mM EDTA, and the volume was reduced to 10
gliters. The sample was subjected to electrophore-
sis on a strip of Whatman 3MM paper for 60 to
80 min at 4 kV with a buffer composed of 5%
acetic acid, 0.5%0 pyridine, 10 mM EDTA, pH
3.5. The paper was dried and cut into 1-cm
squares, and the radioactivity was measured in
toluene-Liquifluor in a Beckman scintillation
counter. Lambda DNA was used as a control in
RF DNA which ranlge ini size from 1,200 to 100
base pairs, in close agreement with the pub-
lished data of Edgell et al. (2).
Chemical evidence for a difference in the recog-
nition site for endonuclease HP and endonuclease
R is presented in Table 2. The nucleotide se-
quence of the recognitioii site for endonuclease
R was determined to be G T Py I Pu A C (4)
where the center dinucleotide is ambiguous and
the arrow indicates the point of cleavage.
We find coinsistent results with qX174 RF DNA
as substrate for endonuclease R (Table 2). The
enzyme preparation had a very low, but detect-
able, level of exonuclease contamination which
might account for the production of 10% pyrimi-
dines. The data indicate that the recognition site
for endonuclease HP is different since only deoxy-
cytidylic acid is found at the 5' ends of frag-
ments produced by this enzyme. The presence of
a single, unique nucleotide at the 5' ends of these
fragments suggests that the recognition site for
endonuclease HP may not contain an ambiguous
dinucleotide as does the site for endonuclease R;
however, we do not regard this demonstration
as rigorous until the terminal nucleotides have
been analyzed for each fragment.
This investigation was supported by Public Health
Service grant GM 13554 from the National Institute of
General Medical Sciences. One of us, P.H.J., is an
American Cancer Society postdoctoral research fellow.
We would like to thank Lawrence I. Grossman for
collaboration in the lambda substrate experiments.
LITERATURE CITED
1. Danna, K., and D. Nathans. 1971. Specific cleavage
of simian virus 40 DNA by restriction endonu-
clease of Hemophilus influenzae. Proc. Nat. Acad.
Sci. U.S.A. 68:2913-2917.
2. Edgell, M. H., C. A. Hutchison, and M. Sclair.
1972. Specific endonuclease R fragments of
bacteriophage *X174 deoxyribonucleic acid. J.
Virol. 9:574-582.
3. Gromkova, R., and S. H. Goodgal. 1972. Action of
Haemophilus endodeoxyribonuclease on bio-
logically active deoxyribonucleic acid. J. Bac-
teriol. 109:987-992.
4. Kelly, T. J., and H. 0. Smith. 1970. A restriction
enzyme from Hemophilue influenzae. II. Base
sequence of the recognition site. J. Mol. Biol.
51:393-409.
5. Loening, U. E. 1967. The fractionation of high
molecular weight ribonucleic acid by polyacryl-
these experiments and was treated identically,
except the reaction with endonucleases was
omitted. The total incorporation of 32P in the
endonuclease HP and endonuclease R reactions
was approximately the same, :1:10%/. Control
experiments were performed without endonu-
clease, and there was no significant incorporation.
b From Kelly and Smith (4).
598 NOTES
VOL. 11, 1973 NOTES
amide gel electrophoresis. Biochem. J. 102:251-
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6. Middleton, J. H., M. H. Edgell, and C. A. Hutchi-
son. 1972. Specific fragments of OX174 deoxy-
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7. Richardson, C. C. 1965. Phosphorylation of nu-
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8. Smith, H. O., and K. W. Wilcox. 1970. A restriction
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cation and general properties. J. Mol. Biol. 51:
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9. Sulkowski, E., and M. Laskowski, Br. 1971. In-
activation of 5'-nucleotidase in commercial
preparations of venom exonuclease (phospho-
diesterase). Biochim. Biophys. Acta 240:443-447.


Production of Specific Fragments of {varphi}X174 Replicative Form DNA by a Restriction Enzyme from Haemophilus parainfluenzae, Endonuclease HP

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Inactivation of 5'-nucleotidase in commercial preparations of venom exonuclease (phosphodiesterase).

Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage599 infected Eacherichia coli.

Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae.

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Production of Specific Fragments of {varphi}X174 Replicative Form DNA by a Restriction Enzyme from Haemophilus parainfluenzae, Endonuclease HP

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