Vol. 91, No. 4, 1979 December
BIOCHEMICAL
RESEARCH COMMUNICATIONS
AND BIOPHYSICAL
28, 1979
Pages
DNA ENDONUCLEASE ACTIVITIES Muriel
Wikswo
Department
Received
ASSOCIATED WITH MELANOMA CELL CHROMATIN
Lambert
of Pathology, Newark,
November
1481-1487
and George
New Jersey Jersey
P. Studzinski
Medical 07102
New
School,
CMDNJ,
16,1979
SUMMARY Chromatin-associated DNA endonucleases, extracted from Cloudman mouse melanoma cell nuclei, were separated on isoelectric focusing into seven in two widely separated groups pH 3.4-5.4 and 7.5-9.3, each fractions active on calf thymus DNA. All fractions in the former group, PI'S 3.4, 4.4 and 5.4, produced at least one single-strand scission per molecule on circular duplex phage PM2 DNA, and transformed circular single-stranded phage fd DNA into linear strands of uniform length. In the second group there was no detectable activity against PM2 DNA, but two fractions PI'S 7.5 and 8.0 were active on fd DNA as above, whereas the other two, ~1's 8.5 and 9.0 transformed fd DNA into a number of different sized, discrete segments. These results indicate that, even allowing for possible enzymatic identity of some of the isoelectrically separated forms, at least three different DNA endonucleases are associated with mouse melanoma cell chromatin.
INTRODUCTION deoxyribonucleases
Classically, gradative
enzymes,
recent
but
number
of
several
DNA endonucleases
different
and
(l-5),
have
may
(7-10). most
cases
nicks
in
not
cell
of
growth
cell
bulk of
which
the cell
these
cells in
DNases
and
been
have
indicated (6)
of these
In
the
enzymes,
mice, to
they
at point
least
and nature
DNA
that
they
and
repair has
in
purified and which produces to
an
here,
enzyme
Cloudman both
facilitate and also
future provide
We have three
a of
propagate
which will enzyme action,
isolation.
enzyme
reported readily
in
reported
however,
may be similar
studies
be de-
purification
DNA replication
because into
contains
isoelectric
mammalian
have recently
and which
relative
for
to
although we have previously chromatin one DNA endonuclease
implanted
parameters
required
differ
as
--et al (11). were chosen
when
cells
preparations
DNA (5),
cells and
chromatin
ions
determined,
Ishida
culture
in
HeLa cell
double-stranded by
studies
location
been
melanoma
these
activities
considered
Isolation
mammalian
on
were
implicates
functions.
from
such
from
described mouse
the
in
The precise
characterized
evidence
cellular
investigations roles
(DNases)
found
that
endonuclease
of activity
towards
in
the
fractDNA.
0006-291X/79/241481-07$01.00/0 1481
Copyright All rights
@ I979
by Academic Press. Inc. in anyform reserved.
of reproduction
Vol. 91, No. 4, 1979
MATERIALS
AND
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
METHODS
Cloudman mouse melanoma cells (S91 NCTC 3960, CCL 53) were propagated by serial subcutaneous injection into male DBA/2J mice. Tumors were harvested, immediately placed at 4’C, minced, passed through gauze, and washed in solution 1 (0.32 M sucrose, 2mM MgCl, 1mM potassium phosphate, pH 6.8). Nuclear isolation was based on the method of Berkowitz al --et (12). Chromatin-associated protein was extracted from the purified nuclei by the method of Fischman et al (5), dialyzed into solution 2 (40% ethylene glycol, 1 mM dithizhrztol, 1 mM Na EDTA , 50 mM potassium phosphate, pH 7.1), passed through a CM Sephadez (Pharmacia) column, and subsequently stored unfrozen at -2OOC. After dialysis against crystalline sucrose at -20°c, the protein solution was electrophoresed on a 110 ml isoelectric focusing column with a 5% to 50% sucrose gradient as described by Fischman et al (11). One ml fractions were collected, their pH determined, a; each fraction assayed for DNA endonuclease activity against calf thymus DNA. Peaks of endonuclease activity were pooled, dialyzed against solution 2, and the protein content of each determined (13). DNA endonuclease activity was assayed using Linear duplex calf thymus DNA (Worthington Biochemical Corp.) as previously described (Lb). This method measures the ability of the enzyme preparation to prime the DNA for DNA polymerase activity. DNA endonuclease activity was $etermined under sterile conditions both against circular single-stranded [ H] phage fd DNA (3.2 n moles P) (Miles Laboratories) and against superhelical doublestranded PM2 (Boehringer Mannheim). The reaction mixture (65 ul) contained enzyme, substrate, 5 mM MgCl 20 mM KCL, and 10 mM Tris-maleate pH 7.5. After 2 hours at 3?‘C, the PHaction was terminated by addition of 10 ul 0.1 M EDTA and 6.5 ul of 10% Sarkosyl (for analysis by neutral 0.7% and 1.4% agarose gels); 15 ul 0.5 M EDTA (for alkaline 1.4% agarose gels); or by the method of Sadowski and Hurwitz (15) for alkaline and neutral sucrose gradients. The samples were then either mixed with 20 ul of 42% sucrose and 0.01% bromophenol blue for analysis on agarose gels or applied directly to sucrose gradients. Agarose gels (6 x 110 mm) were prepared and run, for neutral gels, by the method of Sugden al (16) or, for alkaline gels, by the method of --et Electrophoresis was performed at 5O at either 20 V McDonell al (17). --et for 12 hours (neutral 1.4% gels), 50 V for 3.5 hours (neutral 0.7% gels), or 30 V for 10 hours (alkaline 1.4% gels). Gels with fd DNA were sliced into 1 mm slices on a Gilson Automatic gel fractionator, dissolved in 0.2 ml 60% hydrogen peroxide at 70° overnight and counted in Scintisol (New England Nuclear) in a scintillation counter. Gels with PM2 DNA were stained with ethidium bromide (0.5 ug/mL) and fluorescence of the DNA detected using a C-61 mineralight transilluminator (Ultra-Violet ProDNA was analyzed on 5-20% sucrose gradients by the method of ducts). Sadowski and Hurwitz (15) and radioactivity of each fracticyl determined by DNA exonuclease activity against [ H] poly d(A-T) scintillation counting. (Miles Laboratories) was measured by the method of Lindahl --et al (18).
RESULTS DNA
endonucleases
isoelectric specific indicated
extracted
focusing activity in
Table
into of
each
7
from
mouse
fractions fraction
and on
1.
1482
linear
melanoma
chromatin
into
two
groups
duplex
calf
separate (Fig. thymus
on
1). DNA
The is
BIOCHEMICAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(mll
VOLUME
Isoelectric 1: endonucleases. gradient.
Figure
focusing of mouse e--e, DNase activity
TABLE DNase
activities
associated
melanoma
Total Activitya (units/mg)
cell
chromatin.
3.6
455.6
51.4
126.0
0.012
2
4.4
1040.0
239.6
230.4
0.012
3
5.4
662.4
59.4
89.7
0
4
7.5
395.2
38.2
96.1
Specific Activitya (unitslmg)
Total poly d(A-T)h solubilized
0
48.5
54.1
0
5
8.0
896.4
6
8.5
586.5
107.1
182.8
0
1
9.3
616.4
38.5
62.5
0
The
of on
assay
were
analysis. 2 hours
DNA
for
the
conversion
non-superhelical),
fd
by
resulted Form and
according percentage preparation.
in I to
the
to of
fractions
single-strand
gel
enzymes
amount
1483
in phage
of
the
substrate
of Lindahl hydrolyzed
each
fraction
PM2
DNA and
reaction
electrophoresis
of or
of
to
Form
Form
III
II
each
sucrose
1, 2, and 3 (Table 1) with scissions of the DNA detected
DNA (superhelical) a small
DNA as
the method poly d(A-T)
by
products
subsequent
of
thymus
double-stranded
DNA, and
studied
calf
attack
superhelical
Incubation of
using
endonucleolytic
both
phage
single-stranded
was measured
activity was measured values represent the 80 ug of each enzyme
specificity
examined
gradient
mouse
1
activity
DNA PH
I
Total Protein (ug)
a DNA endonuclease (5). b DNA exonuclease The --et al (18). in 15 minutes by
enzyme
chromatin-associated thymus DNA; O----O,
Isoelectric Point (TO.2)
Fraction
was
with
melanoma on calf
PM2
by
DNA (circular,
DNA (linear,
unit
Vol. 91, No. 4, 1979
Figure
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2: Action of melanoma chromatin-associated endonucleases on PM2 DNA. PM2 DNA (0.5 ug) was incubated for 3 hours at 37’C with the following DNase fractions: a. no enzyme; b. 1 (14Oug) c. 2 (105 ug); d. 3 e. 4 (150 ug); f. 5 (170 up) and applied to 0.7% agarose gels. (75 ug); Gels were stained in ethidium bromide and photographed under ultraviolet light.
3000 2500 2000 DPM 1500 1000 500 0
0 % Rf Figure33:
Electrophoretic profile of r3g] [ H] fd DNA (0.016 uCi) was incubated fraction: a. 2 (30 min.); b. 2 (2 hr.); Gels were sliced into 1 mm slices and determined. Untreated (o---O) and DNase
1484
fd at
DNA on 1.4% agarose gels. 37’C with 90 ug DNase c. 6 (2 hr.); d. 7 (2 hr.). the radioactivity in each was (U) treated DNA.
BIOCHEMICAL
Vol. 91, No. 4, 1979
length)
due
to
cuts
concentration
of Fractions
effect.
across
the
enzyme
or
the 4-7
(Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
double
strand
the
incubation
did
not
1)
(Fig.
2).
time
have
Increasing
did
not
the
change
a detectable
this
effect
on
PM2
each
of
DNA. Treatment fractions
l-5
untreated the
of resulted
DNA,
form
using
of
and
alkaline
on
fd
7
incubation
change
end
of
four
their
incubation Cut
various
cylindrical
DNase
0.7%
agarose
alkaline This
gels.
under
these
incubated Table
confirms
of
that
primarily The
results
gel
all
fd
endonucleases.
to
are
Again,
concentrations At
[3H]
those was
the
retained
fd
seen
DNA
on
indeed
treated on
neutral
single-stranded poly
DNA
was
very
low
d(A-T)
exonuclease
indicates
in
with
characteristics
for
summarized
major
fractions
and
test
This
three
3D).
enzyme
two
incubated
(Fig.
polydeoxynucleotide,
3-7.
study
DNA
additional
into
migration to
at
no
electrophoresis.
fd
in
was
activity
DNA
enzyme
[3H]
activity
fractions
seven
similar
l-7
in
this
higher
the
fractions
DNA of
using
subsequent
synthetic
exonuclease
undetectable
contain
segments
gels that
The
each
1 shows
of
exhibited
agarose
conditions. with
number
was obtained
reaction
had
whereas
Native
fractions
hours
than
and
were
a maximum
four
separated
37’C
cut
The
6
or
DNA.
Circular
been
3C),
on
at
to
faster
results
(Fig. larger
seen
had
analysis.
to
with
migrated
fraction
2 hours
patterns
hours to
the
and
beyond
the
ability
with
DNA
which
Similar
up
electrophoresis
prolonged
not
B).
time
fd
DNA
and
dependent,
a
DNA
gradient
the
into
of
circular
3A
time
treated
gel
separated
did
with
DNA
agarose
fraction
the
sucrose
and
[3H]
peak
(Fig.
Increasing
[3H]
peaks
sharp
that
strands
incubation.
effect.
a
indicating
concentration
hours
single-stranded in
linear
neutral
enzyme
circular
activity.
in
fractions
that
these
Table
was
1 and
2
fractions
2.
DISCUSSION Our mammalian DNA
results
reported
in by
more
other
gest limited activities
that
al
point
cells
indicate
different
there
are of
reported
at cuts
by
least
three
produced recently
to
al
in
of
as
DNAs
fd
other
1485
by
mammalian
the
(19), basis
of
or
one
enzymes
The
however,
present
cell
been
methylation.
substrate,
fractions
of
have nuclei
on
or
enzymes
DNA
this
different
of
a number
whole
separation
acetylation
separate in
in
et
existence
bacteriophage
into
similar
The
deamination,
proteins
focusing
Goodwin (20).
the
by
non-histone
isoelectric somewhat
chromatin
modified
with
number
in
may
on Results
mammalian et
enzymes
experiments
chromatin-associated
separate
activities.
Lambert
isoelectric
that
cells
endonuclease
and
or
show
melanoma
(Table 1-5 systems
is
sug2).
The
similar (1,
to 2,
4,
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
TABLE Summary
of
various
substrates
Group point
1
Group
+ +
+
+
+ or
+ +
+
7.5
4
a.0
+
8.5
+ +
gw3
+ + +
detectable
in
of of
study
The
is
role
DNA
for
these a
that
fraction
4
than
on
DNA
native
is
to
the
DNA
occur
thymus)
(calf
endonucleases
in
active and
thus
5,
are
11).
isolated
similar
However
from
in
chromatin
cells.
mammalian
function
endo-
here
(3, was
whole
nucleotide
restriction
reported
activity from
specific
bacterial
DNA
enzymes
more (23)
with fractions
than
possible
recognize
DNase
endonuclease rather
but
DNA DNA
duplex
enzymes
mammalian
proteins
identified,
in
in
these
known of
other
the
non-histone
incisions incisions
that
Several of
linear
activity
several multiple
as
DNA,
(22). number
on
activity
indicate
may
nucleases
+ + + activity
nucleolytic
Evidence Evidence
sequences
this
+ or
5.4
Detectable
and
a
4.4
show
on
Exonuclease Activity on poly d(A-T)
Single-stranded DNA (fd)
+
fractions No
DNA
+
a All
+ + +
to
Superhelical duplex 0x2)
a
DNases
chromatin-associated
+ + +
3
+ + +
melanoma
+ or
2
-
mouse
2
3.4
Group
b
of
and isoelectric of DNase fraction
Group
211,
activities
RESEARCH COMMUNICATIONS
chromatin
is
suggested
on
partiallly
may
participate
by
has
as
our
recent
depurinated in
repair
fd of
yet
and DNA
not
been
finding PM2
DNA
damage.
Acknowledgements This from
NC1
investigation and
by
Institutional
was
supported funds
by from
NIH ACS
Grants Grant
CA-20043
and
CA-19750
IN-92.
REFERENCES 1. 2. 3. 4.
Wang, E.G., and Furth, J.J. (1977) J. Biol. Chem. 252, 116-124. Brown, F.L., Musich, P.R., and Maio, J.J. (1978) Nucl. Acids Res. 5, 1093-1107. Tang, D. (1978) Nucl. Acids Res. 5, 2861-2875. Watanabe, T., and Kasai, D. (1978) Biochim. Biophys. Acta 520, 52-60.
1486
Vol. 91, No. 4, 1979
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fischman, WatanabeG...T., Lambert, M.W., and Studzinski, G.P. (1979) Biochim. Biophys. Acta Brown567, 464-471. Ionnou, P. (1973) Nature-New Biol. 244, 257-260. T.P. (1976) Biochim. Biophys. Acta 454, 172-183. Brent, Cleaver, J.E. (1978) Biochim. Biophys. Acta 516, 489-516. Nes, I.F., and Nissen-Meyer, J. (1978) Biochim. Biophys. Acta 520, 111-121. Lindahl, T. (1979) Prog. Nucl. Acid Res. 22, 135-192. Ishida, R., Akiyoshi, H., and Takahashi, T. (1974) Biochem. Biophys. Res. Comm. 56, 703-710. Berkowitz, D.M., Kakefuda, T., and Sporn, M.B.A. (1969) .I Cell Bio1.42, 851-855. Lowry, G.H., Rosenbrough, N.J., Farr, A.L., and Randal, R.J. (1951) J. Biol Chem. 193, 265-275. Studzinski, G.P., and Fischman, G.T. (1974) Anal. Biochem. 58, 449-458. Sadowski, P.D., and Hurwitz, J. (1969) J. Biol. Chem. 244, 6182-6191. Sugden, B., DeTroy, B., Roberts, R.J., and Sambrook, J. (1975) Anal. Biochem. 68, 36-46. McDonell, M.W., Simon, M.N., and Studier, F.W. (1977) J. Mol. Biol. 110, 119-146. Lindahl, T., Gally, J.A., and Edelman, G. M. (1969) Proc. Natl. Acad. Sci. U.S.A. 62, 597-603. Goodwin, R.G., Sei, K.S., and Karu, A.E. (1978) Fed. Proc. 37, 1413. Lambert, W.C., Kolber, L. Okorodudu, A., and Lambert, M.W. (1979) Fed. Proc. 38, 995. Lavin, M.F., Kikuchi, T., Counsilman, C., Jenkins, A., Winzor, D.J., and Kidson, C. (1976) Biochemistry 15, 2409-2414. Roberts, R.J. (1978) Gene 4, 183-193. Lambert, M.W., and Studzinski, G.P. (1979) Fed. Proc. 38, 994.
1487
BIOCHEMICAL
RESEARCH COMMUNICATIONS
AND BIOPHYSICAL
28, 1979
Pages
DNA ENDONUCLEASE ACTIVITIES Muriel
Wikswo
Department
Received
ASSOCIATED WITH MELANOMA CELL CHROMATIN
Lambert
of Pathology, Newark,
November
1481-1487
and George
New Jersey Jersey
P. Studzinski
Medical 07102
New
School,
CMDNJ,
16,1979
SUMMARY Chromatin-associated DNA endonucleases, extracted from Cloudman mouse melanoma cell nuclei, were separated on isoelectric focusing into seven in two widely separated groups pH 3.4-5.4 and 7.5-9.3, each fractions active on calf thymus DNA. All fractions in the former group, PI'S 3.4, 4.4 and 5.4, produced at least one single-strand scission per molecule on circular duplex phage PM2 DNA, and transformed circular single-stranded phage fd DNA into linear strands of uniform length. In the second group there was no detectable activity against PM2 DNA, but two fractions PI'S 7.5 and 8.0 were active on fd DNA as above, whereas the other two, ~1's 8.5 and 9.0 transformed fd DNA into a number of different sized, discrete segments. These results indicate that, even allowing for possible enzymatic identity of some of the isoelectrically separated forms, at least three different DNA endonucleases are associated with mouse melanoma cell chromatin.
INTRODUCTION deoxyribonucleases
Classically, gradative
enzymes,
recent
but
number
of
several
DNA endonucleases
different
and
(l-5),
have
may
(7-10). most
cases
nicks
in
not
cell
of
growth
cell
bulk of
which
the cell
these
cells in
DNases
and
been
have
indicated (6)
of these
In
the
enzymes,
mice, to
they
at point
least
and nature
DNA
that
they
and
repair has
in
purified and which produces to
an
here,
enzyme
Cloudman both
facilitate and also
future provide
We have three
a of
propagate
which will enzyme action,
isolation.
enzyme
reported readily
in
reported
however,
may be similar
studies
be de-
purification
DNA replication
because into
contains
isoelectric
mammalian
have recently
and which
relative
for
to
although we have previously chromatin one DNA endonuclease
implanted
parameters
required
differ
as
--et al (11). were chosen
when
cells
preparations
DNA (5),
cells and
chromatin
ions
determined,
Ishida
culture
in
HeLa cell
double-stranded by
studies
location
been
melanoma
these
activities
considered
Isolation
mammalian
on
were
implicates
functions.
from
such
from
described mouse
the
in
The precise
characterized
evidence
cellular
investigations roles
(DNases)
found
that
endonuclease
of activity
towards
in
the
fractDNA.
0006-291X/79/241481-07$01.00/0 1481
Copyright All rights
@ I979
by Academic Press. Inc. in anyform reserved.
of reproduction
Vol. 91, No. 4, 1979
MATERIALS
AND
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
METHODS
Cloudman mouse melanoma cells (S91 NCTC 3960, CCL 53) were propagated by serial subcutaneous injection into male DBA/2J mice. Tumors were harvested, immediately placed at 4’C, minced, passed through gauze, and washed in solution 1 (0.32 M sucrose, 2mM MgCl, 1mM potassium phosphate, pH 6.8). Nuclear isolation was based on the method of Berkowitz al --et (12). Chromatin-associated protein was extracted from the purified nuclei by the method of Fischman et al (5), dialyzed into solution 2 (40% ethylene glycol, 1 mM dithizhrztol, 1 mM Na EDTA , 50 mM potassium phosphate, pH 7.1), passed through a CM Sephadez (Pharmacia) column, and subsequently stored unfrozen at -2OOC. After dialysis against crystalline sucrose at -20°c, the protein solution was electrophoresed on a 110 ml isoelectric focusing column with a 5% to 50% sucrose gradient as described by Fischman et al (11). One ml fractions were collected, their pH determined, a; each fraction assayed for DNA endonuclease activity against calf thymus DNA. Peaks of endonuclease activity were pooled, dialyzed against solution 2, and the protein content of each determined (13). DNA endonuclease activity was assayed using Linear duplex calf thymus DNA (Worthington Biochemical Corp.) as previously described (Lb). This method measures the ability of the enzyme preparation to prime the DNA for DNA polymerase activity. DNA endonuclease activity was $etermined under sterile conditions both against circular single-stranded [ H] phage fd DNA (3.2 n moles P) (Miles Laboratories) and against superhelical doublestranded PM2 (Boehringer Mannheim). The reaction mixture (65 ul) contained enzyme, substrate, 5 mM MgCl 20 mM KCL, and 10 mM Tris-maleate pH 7.5. After 2 hours at 3?‘C, the PHaction was terminated by addition of 10 ul 0.1 M EDTA and 6.5 ul of 10% Sarkosyl (for analysis by neutral 0.7% and 1.4% agarose gels); 15 ul 0.5 M EDTA (for alkaline 1.4% agarose gels); or by the method of Sadowski and Hurwitz (15) for alkaline and neutral sucrose gradients. The samples were then either mixed with 20 ul of 42% sucrose and 0.01% bromophenol blue for analysis on agarose gels or applied directly to sucrose gradients. Agarose gels (6 x 110 mm) were prepared and run, for neutral gels, by the method of Sugden al (16) or, for alkaline gels, by the method of --et Electrophoresis was performed at 5O at either 20 V McDonell al (17). --et for 12 hours (neutral 1.4% gels), 50 V for 3.5 hours (neutral 0.7% gels), or 30 V for 10 hours (alkaline 1.4% gels). Gels with fd DNA were sliced into 1 mm slices on a Gilson Automatic gel fractionator, dissolved in 0.2 ml 60% hydrogen peroxide at 70° overnight and counted in Scintisol (New England Nuclear) in a scintillation counter. Gels with PM2 DNA were stained with ethidium bromide (0.5 ug/mL) and fluorescence of the DNA detected using a C-61 mineralight transilluminator (Ultra-Violet ProDNA was analyzed on 5-20% sucrose gradients by the method of ducts). Sadowski and Hurwitz (15) and radioactivity of each fracticyl determined by DNA exonuclease activity against [ H] poly d(A-T) scintillation counting. (Miles Laboratories) was measured by the method of Lindahl --et al (18).
RESULTS DNA
endonucleases
isoelectric specific indicated
extracted
focusing activity in
Table
into of
each
7
from
mouse
fractions fraction
and on
1.
1482
linear
melanoma
chromatin
into
two
groups
duplex
calf
separate (Fig. thymus
on
1). DNA
The is
BIOCHEMICAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(mll
VOLUME
Isoelectric 1: endonucleases. gradient.
Figure
focusing of mouse e--e, DNase activity
TABLE DNase
activities
associated
melanoma
Total Activitya (units/mg)
cell
chromatin.
3.6
455.6
51.4
126.0
0.012
2
4.4
1040.0
239.6
230.4
0.012
3
5.4
662.4
59.4
89.7
0
4
7.5
395.2
38.2
96.1
Specific Activitya (unitslmg)
Total poly d(A-T)h solubilized
0
48.5
54.1
0
5
8.0
896.4
6
8.5
586.5
107.1
182.8
0
1
9.3
616.4
38.5
62.5
0
The
of on
assay
were
analysis. 2 hours
DNA
for
the
conversion
non-superhelical),
fd
by
resulted Form and
according percentage preparation.
in I to
the
to of
fractions
single-strand
gel
enzymes
amount
1483
in phage
of
the
substrate
of Lindahl hydrolyzed
each
fraction
PM2
DNA and
reaction
electrophoresis
of or
of
to
Form
Form
III
II
each
sucrose
1, 2, and 3 (Table 1) with scissions of the DNA detected
DNA (superhelical) a small
DNA as
the method poly d(A-T)
by
products
subsequent
of
thymus
double-stranded
DNA, and
studied
calf
attack
superhelical
Incubation of
using
endonucleolytic
both
phage
single-stranded
was measured
activity was measured values represent the 80 ug of each enzyme
specificity
examined
gradient
mouse
1
activity
DNA PH
I
Total Protein (ug)
a DNA endonuclease (5). b DNA exonuclease The --et al (18). in 15 minutes by
enzyme
chromatin-associated thymus DNA; O----O,
Isoelectric Point (TO.2)
Fraction
was
with
melanoma on calf
PM2
by
DNA (circular,
DNA (linear,
unit
Vol. 91, No. 4, 1979
Figure
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2: Action of melanoma chromatin-associated endonucleases on PM2 DNA. PM2 DNA (0.5 ug) was incubated for 3 hours at 37’C with the following DNase fractions: a. no enzyme; b. 1 (14Oug) c. 2 (105 ug); d. 3 e. 4 (150 ug); f. 5 (170 up) and applied to 0.7% agarose gels. (75 ug); Gels were stained in ethidium bromide and photographed under ultraviolet light.
3000 2500 2000 DPM 1500 1000 500 0
0 % Rf Figure33:
Electrophoretic profile of r3g] [ H] fd DNA (0.016 uCi) was incubated fraction: a. 2 (30 min.); b. 2 (2 hr.); Gels were sliced into 1 mm slices and determined. Untreated (o---O) and DNase
1484
fd at
DNA on 1.4% agarose gels. 37’C with 90 ug DNase c. 6 (2 hr.); d. 7 (2 hr.). the radioactivity in each was (U) treated DNA.
BIOCHEMICAL
Vol. 91, No. 4, 1979
length)
due
to
cuts
concentration
of Fractions
effect.
across
the
enzyme
or
the 4-7
(Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
double
strand
the
incubation
did
not
1)
(Fig.
2).
time
have
Increasing
did
not
the
change
a detectable
this
effect
on
PM2
each
of
DNA. Treatment fractions
l-5
untreated the
of resulted
DNA,
form
using
of
and
alkaline
on
fd
7
incubation
change
end
of
four
their
incubation Cut
various
cylindrical
DNase
0.7%
agarose
alkaline This
gels.
under
these
incubated Table
confirms
of
that
primarily The
results
gel
all
fd
endonucleases.
to
are
Again,
concentrations At
[3H]
those was
the
retained
fd
seen
DNA
on
indeed
treated on
neutral
single-stranded poly
DNA
was
very
low
d(A-T)
exonuclease
indicates
in
with
characteristics
for
summarized
major
fractions
and
test
This
three
3D).
enzyme
two
incubated
(Fig.
polydeoxynucleotide,
3-7.
study
DNA
additional
into
migration to
at
no
electrophoresis.
fd
in
was
activity
DNA
enzyme
[3H]
activity
fractions
seven
similar
l-7
in
this
higher
the
fractions
DNA of
using
subsequent
synthetic
exonuclease
undetectable
contain
segments
gels that
The
each
1 shows
of
exhibited
agarose
conditions. with
number
was obtained
reaction
had
whereas
Native
fractions
hours
than
and
were
a maximum
four
separated
37’C
cut
The
6
or
DNA.
Circular
been
3C),
on
at
to
faster
results
(Fig. larger
seen
had
analysis.
to
with
migrated
fraction
2 hours
patterns
hours to
the
and
beyond
the
ability
with
DNA
which
Similar
up
electrophoresis
prolonged
not
B).
time
fd
DNA
and
dependent,
a
DNA
gradient
the
into
of
circular
3A
time
treated
gel
separated
did
with
DNA
agarose
fraction
the
sucrose
and
[3H]
peak
(Fig.
Increasing
[3H]
peaks
sharp
that
strands
incubation.
effect.
a
indicating
concentration
hours
single-stranded in
linear
neutral
enzyme
circular
activity.
in
fractions
that
these
Table
was
1 and
2
fractions
2.
DISCUSSION Our mammalian DNA
results
reported
in by
more
other
gest limited activities
that
al
point
cells
indicate
different
there
are of
reported
at cuts
by
least
three
produced recently
to
al
in
of
as
DNAs
fd
other
1485
by
mammalian
the
(19), basis
of
or
one
enzymes
The
however,
present
cell
been
methylation.
substrate,
fractions
of
have nuclei
on
or
enzymes
DNA
this
different
of
a number
whole
separation
acetylation
separate in
in
et
existence
bacteriophage
into
similar
The
deamination,
proteins
focusing
Goodwin (20).
the
by
non-histone
isoelectric somewhat
chromatin
modified
with
number
in
may
on Results
mammalian et
enzymes
experiments
chromatin-associated
separate
activities.
Lambert
isoelectric
that
cells
endonuclease
and
or
show
melanoma
(Table 1-5 systems
is
sug2).
The
similar (1,
to 2,
4,
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
TABLE Summary
of
various
substrates
Group point
1
Group
+ +
+
+
+ or
+ +
+
7.5
4
a.0
+
8.5
+ +
gw3
+ + +
detectable
in
of of
study
The
is
role
DNA
for
these a
that
fraction
4
than
on
DNA
native
is
to
the
DNA
occur
thymus)
(calf
endonucleases
in
active and
thus
5,
are
11).
isolated
similar
However
from
in
chromatin
cells.
mammalian
function
endo-
here
(3, was
whole
nucleotide
restriction
reported
activity from
specific
bacterial
DNA
enzymes
more (23)
with fractions
than
possible
recognize
DNase
endonuclease rather
but
DNA DNA
duplex
enzymes
mammalian
proteins
identified,
in
in
these
known of
other
the
non-histone
incisions incisions
that
Several of
linear
activity
several multiple
as
DNA,
(22). number
on
activity
indicate
may
nucleases
+ + + activity
nucleolytic
Evidence Evidence
sequences
this
+ or
5.4
Detectable
and
a
4.4
show
on
Exonuclease Activity on poly d(A-T)
Single-stranded DNA (fd)
+
fractions No
DNA
+
a All
+ + +
to
Superhelical duplex 0x2)
a
DNases
chromatin-associated
+ + +
3
+ + +
melanoma
+ or
2
-
mouse
2
3.4
Group
b
of
and isoelectric of DNase fraction
Group
211,
activities
RESEARCH COMMUNICATIONS
chromatin
is
suggested
on
partiallly
may
participate
by
has
as
our
recent
depurinated in
repair
fd of
yet
and DNA
not
been
finding PM2
DNA
damage.
Acknowledgements This from
NC1
investigation and
by
Institutional
was
supported funds
by from
NIH ACS
Grants Grant
CA-20043
and
CA-19750
IN-92.
REFERENCES 1. 2. 3. 4.
Wang, E.G., and Furth, J.J. (1977) J. Biol. Chem. 252, 116-124. Brown, F.L., Musich, P.R., and Maio, J.J. (1978) Nucl. Acids Res. 5, 1093-1107. Tang, D. (1978) Nucl. Acids Res. 5, 2861-2875. Watanabe, T., and Kasai, D. (1978) Biochim. Biophys. Acta 520, 52-60.
1486
Vol. 91, No. 4, 1979
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fischman, WatanabeG...T., Lambert, M.W., and Studzinski, G.P. (1979) Biochim. Biophys. Acta Brown567, 464-471. Ionnou, P. (1973) Nature-New Biol. 244, 257-260. T.P. (1976) Biochim. Biophys. Acta 454, 172-183. Brent, Cleaver, J.E. (1978) Biochim. Biophys. Acta 516, 489-516. Nes, I.F., and Nissen-Meyer, J. (1978) Biochim. Biophys. Acta 520, 111-121. Lindahl, T. (1979) Prog. Nucl. Acid Res. 22, 135-192. Ishida, R., Akiyoshi, H., and Takahashi, T. (1974) Biochem. Biophys. Res. Comm. 56, 703-710. Berkowitz, D.M., Kakefuda, T., and Sporn, M.B.A. (1969) .I Cell Bio1.42, 851-855. Lowry, G.H., Rosenbrough, N.J., Farr, A.L., and Randal, R.J. (1951) J. Biol Chem. 193, 265-275. Studzinski, G.P., and Fischman, G.T. (1974) Anal. Biochem. 58, 449-458. Sadowski, P.D., and Hurwitz, J. (1969) J. Biol. Chem. 244, 6182-6191. Sugden, B., DeTroy, B., Roberts, R.J., and Sambrook, J. (1975) Anal. Biochem. 68, 36-46. McDonell, M.W., Simon, M.N., and Studier, F.W. (1977) J. Mol. Biol. 110, 119-146. Lindahl, T., Gally, J.A., and Edelman, G. M. (1969) Proc. Natl. Acad. Sci. U.S.A. 62, 597-603. Goodwin, R.G., Sei, K.S., and Karu, A.E. (1978) Fed. Proc. 37, 1413. Lambert, W.C., Kolber, L. Okorodudu, A., and Lambert, M.W. (1979) Fed. Proc. 38, 995. Lavin, M.F., Kikuchi, T., Counsilman, C., Jenkins, A., Winzor, D.J., and Kidson, C. (1976) Biochemistry 15, 2409-2414. Roberts, R.J. (1978) Gene 4, 183-193. Lambert, M.W., and Studzinski, G.P. (1979) Fed. Proc. 38, 994.
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