(Received for publication, March 3, 1995; and in revised form, June 6, 1995)
From the
Small cell lung cancer cells (OC-NYH-VM) were permeabilized and
treated with different nucleases. The long-range distribution of DNA
cleavage sites in the amplified c-myc gene locus was then
analyzed by pulsed field gel electrophoretic separation of the released
50-kilobase to 1-megabase DNA fragments followed by indirect end
labeling. Exogenous DNase I and nucleases specific for the
single-stranded DNA were found to generate similar nonrandom patterns
of large DNA fragments. The cleavage sites were located close to or
even colocalized with matrix attachment regions, which were mapped
independently using a recently developed procedure for DNA loop
excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous
acidic nuclease with the properties of DNase II also digested DNA
preferentially in proximity to the matrix attachment regions,
generating characteristic patterns of excised DNA loops and their
oligomers. A similar, although less specific, pattern of DNA
fragmentation was observed after incubation of permeabilized cells
under conditions favoring the activity of endogenous neutral
Ca
It has been shown previously that treatment of metaphase
chromosomes with Bal-31 nuclease results in the excision of large DNA
fragments ranging in size from 50 to 300 kb( Surprisingly, most of these enzymes, with the exception of
micrococcal nuclease, were found to cleave nuclear DNA in permeabilized
cells preferentially in proximity to the matrix attachment regions,
thus generating the same sets of large DNA fragments as those generated
by topoisomerase II-mediated cleavage. Micrococcal nuclease showed
little specificity of DNA fragmentation. No specific fragmentation of
the amplified c-myc gene locus was observed when the nuclei
with disrupted chromatin structure or naked DNA were treated with
exogenous nucleases. Hence, the preferential sensitivity of matrix
attachment regions to nuclease digestion is determined by DNA
interaction with nuclear proteins.
Human small cell lung cancer cells embedded in agarose blocks were
placed in the relevant buffer supplemented with 0.1% Nonidet P-40 (for
cell permeabilization) and were incubated at 37 °C for different
periods of time as described in the figure legends. In buffer A,
gradual degradation of genomic DNA into large fragments ranging in size
from 50 kb to 1 megabase was observed (Fig. 1A, leftpanel). The size distribution of the released
fragments depended on the time of incubation, with shorter fragments
being more abundant after longer times of incubation. Digestion of
nuclear DNA by endogenous nucleases was clearly inhibited by the
addition of 2 mM Zn
Figure 1:
Fragments of genomic DNA released by
endogenous nucleases. A, incubation of permeabilized cells in
neutral buffer supplemented with Ca
In contrast to the
results of other studies(2, 13) , we failed to observe
any discontinuity in the size distribution of the released DNA
fragments when PFGE was used. However, discontinuity was observed when
field inversion gel electrophoresis was used for separation of the same
DNA samples (data not shown). Thus, it seems possible that the discrete
300-kb band observed previously by several investigators (2, 3, 13) is a compression artifact of field
inversion gel electrophoresis used for separation of the excised DNA
fragments. Indeed, in no case did these authors examine the separation
of phage
Figure 2:
Size distribution of DNA fragments
released by treatment of permeabilized cells with exogenous nucleases. A, cells were treated for 10 min at 37 °C in 1 ml of
buffer B with 60, 150, and 300 units of mung bean nuclease (lanes
2-4, respectively) or with 150 and 300 units of S1 nuclease (lanes5 and 6, respectively). Lane1 represents DNA from cells incubated in buffer B without
enzyme. Positions of markers (phage
A gradual degradation of DNA similar to that
seen at pH 7.5 was observed when permeabilized cells were incubated in
buffers B and C (Fig. 1B). It is important to note that no
significant degradation of nuclear DNA by endogenous nucleases was
observed in our experiments at short incubation times of 10 min at pH
5.0, irrespective of the presence of divalent cations (Fig. 1B), or up to 30 min at pH 7.5 in the presence of
Ca
Figure 3:
Mapping of DNA cleavages downstream from
the c-myc gene generated by endogenous acidic nuclease (B) and by single-stranded DNA-specific nucleases (C). A, shown is a map of the c-myc amplicon
organization in OC-NYH-VM cells. The positions of the gene are shown as openrectangles. The third exon of the c-myc gene, which was used as a probe in hybridization experiments, is
located just downstream of the pair of NotI cleavage sites
indicated by arrows. B, permeabilized cells were
incubated for 20 min (lanes3 and 4) or 40
min (lanes2 and 5) in pH 5.0 buffer free of
divalent cations (buffer C (lanes2 and 3))
or supplemented with Zn
Incubation of permeabilized cells at
37 °C in pH 5.0 buffer (buffers B and C) resulted in the
accumulation of fragments of 80, 150, and 260 kb (Fig. 3B, lanes 2-5). Essentially the
same fragments were excised from the c-myc amplicon by
topoisomerase II-mediated DNA cleavage either in vivo or after
the disruption of chromatin structure by 2 M salt extraction (Fig. 3B, lanes 6 and 7,
respectively). The similarity in the patterns of c-myc amplicon fragmentation by nuclear matrix-bound topoisomerase II
and by endogenous acidic nucleases (Fig. 3B, compare lane7 with lanes3 and 5)
suggests that matrix attachment regions may constitute preferential
targets for endogenous acidic nucleases. The activity of acidic
nucleases did not depend on the presence of Mg In the next set of experiments, the
specificity of c-myc amplicon fragmentation by exogenous
single-stranded DNA-specific nucleases was studied. Fig. 3C shows the patterns of large DNA fragments excised from the
c-myc amplicon by S1 and mung bean nucleases. Again, the
patterns of released fragments were very similar to that resulting from
DNA cleavage by matrix-bound topoisomerase II (Fig. 3C, lane5), suggesting that both enzymes preferentially
attack nuclear DNA at matrix attachment regions. Although treatments
with both mung bean and S1 nucleases were carried out under conditions
favorable for the activity of endogenous acidic nucleases, the time of
incubation was too short for this activity to contribute significantly
to the resulting cleavage pattern. Indeed, no specific fragmentation of
DNA was evident when permeabilized cells were incubated in the
digestion buffer without exogenous enzymes (Fig. 3C, lane7). Furthermore, the accumulation of specific
cleavage products was observed when the concentration of exogenous
nuclease was increased (Fig. 3C), indicating that the
appearance of specific 80-, 150-, and 260-kb fragments is due to the
activity of exogenous nuclease. These results are in agreement with
previous observations that indicate the possibility of excision of
chromosomal DNA loops by Bal-31 (1) and S1 (15) nucleases. The characteristic 80-, 150-, and 260-kb
bands, similar to those generated by topoisomerase II-mediated cleavage
at matrix attachment sites, could also be recognized among the cleavage
products generated by the endogenous neutral Ca
Figure 4:
Mapping of DNA cleavages downstream from
the c-myc gene generated by endogenous neutral
Ca
Having characterized the
pattern of cleavages within the c-myc amplicon by endogenous
neutral Ca
Figure 5:
A, pattern of DNase I cleavage in high
salt-extracted nuclei. Treatment of nuclei pre-extracted with 2 M NaCl was carried out for 30 min at 0 °C with 0, 2, 4, 6, and 8
µg of DNase I (lanes 1-5, respectively) in 1 ml of
buffer A. After separation by PFGE and Southern transfer, fragments
bearing the third exon of the c-myc gene were visualized by
hybridization. B, fragmentation of partially denatured DNA by
S1 nuclease. Naked DNA was treated with 10, 25, 50, and 150 units of S1
nuclease (lanes 2-5, respectively) in buffer B
containing 50% formamide. After separation by PFGE and Southern
transfer, fragments bearing the third exon of the c-myc gene
were visualized by hybridization. Lane 1 demonstrates the
distribution of DNA products of endogenous topoisomerase II-mediated
cleavage in vivo.
Micrococcal nuclease produced an almost
random pattern of large fragments of the c-myc amplicon, in
contrast to all the other nucleases studied (Fig. 4C),
although preferential cleavage 80 kb downstream from the c-myc gene was still observed. This less specific pattern may be
explained at least in part by the small size (12 kDa) of this enzyme as
compared with 31 kDa for DNase I. It may happen that chromatin
structure imposes stronger restrictions on the accessibility for larger
enzymes. Indeed, it is known that the hypersensitive sites for DNase I
and micrococcal nuclease frequently do not coincide when mapped with
higher resolution (16) , and hence, it is not surprising that
the patterns of long-range DNA cleavage by these two enzymes have only
a limited degree of similarity.
The specificity of cleavage of nuclear DNA by different
nucleases has been intensively studied since the first observation of
preferential digestion of active genes by exogenous DNase
I(19) . These studies made it possible to identify
nuclease-sensitive genomic domains and also hypersensitive sites in
chromatin, which are usually considered as nucleosome-free
regions(16) . At hypersensitive sites, the DNA may also be
preferentially accessible to different trans-acting factors,
and hence, it is not surprising that these sites are frequently found
within the regulatory regions of genes and gene clusters. The
distribution of DNase I-hypersensitive sites, which also constitute
preferential targets for certain other exogenous nucleases, has been
studied in a number of genomic areas by high resolution
mapping(16) . Here we describe a first attempt to
characterize the long-range specificity of nuclear DNA cleavage by
endogenous and exogenous nucleases. Our main finding is that the
long-range distribution of preferential targets for different nucleases
in chromatin is not random. In other words, the long-range DNA
fragmentation by most of the nucleases tested proceeds via preferential
cleavages at distinct genomic positions. In the amplified human
c-myc gene locus, the cleavage regions are located at more or
less regular intervals and delimit less accessible domains of
80-110 kb in length. Hence, it is likely that the distribution of
cleavage regions reflects some periodicity of higher order DNA
packaging in cell nuclei. The only known structural units of chromatin
that could be related to this periodicity are DNA loops attached to the
nuclear matrix (see Refs. 20 and 21 for a review). Studies of the size
distribution of large DNA fragments cleaved from genomic DNA by
endogenous nucleases (2) and by Bal-31 nuclease (1) led
to the suggestion that each loop possesses one preferential target for
these nucleases. Our data support this suggestion and furthermore
indicate that the above-mentioned target is located close to the
regions of DNA loop anchorage to the nuclear matrix or even within
these regions. This conclusion follows from a comparison of the
distribution of nuclease-sensitive regions with the regions of DNA
anchored to the nuclear matrix, which we have mapped by topoisomerase
II-mediated DNA cleavage, as described earlier(7) . Different
nucleases show various degrees of preference for cleavage at or close
to the matrix attachment regions. Endogenous acidic nucleases generate
a pattern of large DNA cleavage products that is very similar to those
produced by matrix-bound topoisomerase II. However, the actual cutting
sites for the different nucleases may not be identical since the level
of error in fragment sizing could be several kilobases under the
conditions that have been used. The same patterns are also generated by
S1 and mung bean nucleases, indicating that the regular secondary
structure of DNA may be modified at matrix attachment sites. These
results are in good agreement with our previous observations made on
the domain of chicken Our data have an important biological implication:
the sites of attachment of DNA loops to the nuclear matrix constitute
weak points in chromatin or chromatids. The preferential accessibility
of these regions to nucleases could increase the probability of
occasional DNA breaks within matrix attachment regions. Such breaks are
believed to promote events resulting in DNA deletion, translocation,
and/or amplification(23) . Hence, it is likely that matrix
attachment sites may constitute hot spots of chromosomal DNA
rearrangement(24) . Interestingly, the distances between
recombination hot spots mapped within several genomic regions
correspond approximately to the size of DNA
loops(25, 26, 27) . Furthermore, the
degenerate sequence organization of matrix-bound DNA(28) ,
together with the localization of at least part of topoisomerase II at
the nuclear
matrix(29, 30, 31, 32, 33) ,
may promote illegitimate recombination between loop ends.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and Mg
-dependent nucleases.
These findings are discussed in the context of the current model of the
spatial domain organization of eukaryotic genome.
)(1) .
Similar size products also accumulate at initial steps of apoptosis, i.e. when chromosomal DNA is degraded by endogenous
nucleases(2, 3, 4, 5) . It has been
suggested that the pattern of large-scale fragmentation of chromosomal
DNA by either exogenous or endogenous nucleases reflects the
organization of the 30-nm chromatin fiber into loops attached to the
nuclear matrix(2, 3, 5, 6) . This
assumption is only supported, however, by a certain similarity in
fragment lengths. To gain further insights into the mechanisms by which
large-scale fragmentation of the eukaryotic genome takes place, it
seemed important to study the specificity of such fragmentation, i.e. to find out if it proceeds via preferential DNA cleavage
at distinct places in the genome. This type of analysis also provides
the possibility to check whether the large DNA fragments excised from
the genome by nuclease-mediated cleavage are indeed the chromosomal DNA
loops. Recently, we have developed a new experimental technique that
allows DNA loops to be excised by topoisomerase II-mediated DNA
cleavage at matrix attachment sites(7, 8) . Separation
of the excised DNA fragments by pulsed field gel electrophoresis (9) followed by indirect end labeling of fragments bearing
different probes made it possible to study the DNA loop organization
within an amplified human c-myc gene locus(7) . In the
present work, we have used the same experimental system and mapping
approach to determine whether fragmentation of nuclear DNA by
endogenous and exogenous nucleases proceeds in a specific way and, if
so, how this specificity correlates with the organization of genomic
DNA into loops. With this aim, the patterns of fragmentation of the
amplified c-myc gene locus by exogenous DNase I, micrococcal
nuclease, mung bean nuclease, S1 nuclease, endogenous
Ca
- and Mg
-dependent nucleases, and
endogenous acidic nucleases were compared with the pattern of
topoisomerase II-mediated fragmentation of the same genomic region.
Cells
The human small cell lung cancer line
OC-NYH-VM (10) was maintained in RPMI 1640 medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum, 100
units/ml penicillin, 100 µg/ml streptomycin, and 2 mM sodium glutamate.Embedding of Cells in Agarose Blocks
Growing cells
were pelleted by centrifugation and washed once with RPMI 1640 medium.
Approximately 4 10
pelleted cells were resuspended
in 1 ml of RPMI 1640 medium (preheated to 37 °C) and mixed with an
equal volume of molten 1.5% (w/v) low-melting temperature agarose
(Bio-Rad) prepared in RPMI 1640 medium. The suspension was distributed
into 10 molds (20
9
1.2 mm) and left at room
temperature for 5 min. Each agarose block thus contained
4
10
cells. The block's thickness allowed efficient
diffusion of drugs and enzymes during treatment of cells or naked DNA
samples.
Treatment of Cells with VM-26
To induce
accumulation of intermediate DNA-topoisomerase II cleavable complexes,
the agarose blocks with cells were incubated for 30 min at 37 °C in
RPMI 1640 medium supplemented with 40 µM VM-26
(4`-demethylepipodophyllotoxin--D-thenylidene glucoside),
an inhibitor of the breakage-reunion reaction catalyzed by
topoisomerase II(11) . After incubation, the blocks were
transferred into stop buffer containing 0.2 M NaEDTA (pH 8.0),
1% SDS, and 1 mg/ml proteinase K (Merck). Protein digestion was
performed for 36 h at 50 °C with constant rotation. The agarose
blocks were then washed with 0.2 M NaEDTA (pH 8.0) and stored
in this solution at 4 °C.
Two molar NaCl Extraction of Permeabilized Cells and
Treatment with VM-26 or DNase I
To permeabilize cells and to
extract them with 2 M NaCl, the agarose blocks with embedded
cells were incubated in solution containing 0.1% Nonidet P-40, 2 M NaCl, 2 mM NaEDTA, 1 mM phenylmethylsulfonyl
fluoride, and 20 mM Tris-HCl (pH 7.5). Incubation was carried
out for 1 h at 4 °C. The blocks were then washed three times (30
min each) at 4 °C in 20 mM Tris-HCl (pH 7.5), 0.1 mM NaEDTA, 50 mM KCl, and 10 mM MgCl (topoisomerase II cleavage buffer) supplemented with the
following protein inhibitors: pepstatin at 1 µg/ml, aprotinin at 5
µg/ml, and leupeptin at 1 µg/ml. After the washing step, the
blocks were placed in the same solution supplemented with 40 µM VM-26 and incubated first for 10 min at 4 °C and then for 40
min at 37 °C. Subsequently, the topoisomerase II reaction was
stopped by transferring the agarose blocks into stop buffer, and
proteins were digested as described above. DNase I treatment of nuclei
pre-extracted with 2 M NaCl was carried out for 30 min at 0
°C in buffer A (10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1.5 mM MgCl
, and 1.5 mM CaCl
) supplemented with different amounts of the
enzyme. The reactions were terminated as described above.
Treatment of Permeabilized Cells with
Nucleases
Cells embedded in agarose blocks were permeabilized by
incubation of the blocks in one of the following solutions to which
Nonidet P-40 had been added to 0.1%: buffer A, buffer B (30 mM sodium acetate (pH 5.0), 50 mM NaCl, and 2 mM ZnCl), or buffer C (30 mM sodium acetate (pH
5.0), 50 mM NaCl, and 2 mM NaEDTA). The activity of
the endogenous acidic nucleases was studied by incubation of embedded
cells in buffer B or C at 37 °C for different periods of time as
specified in the figure legends. Fragmentation of nuclear DNA by
endogenous neutral Ca
- and
Mg
-dependent nucleases was studied in a similar way,
except that buffer A was used. In some experiments, 2 mM ZnCl
was added to buffer A. Treatment of permeabilized
cells with DNase I and micrococcal nuclease was carried out for 30 min
at 0 °C in buffer A supplemented with different amounts of the
above enzymes as specified in the figure legends. Treatment of
permeabilized cells with S1 and mung bean nucleases was carried out in
buffer B for 10 min at 37 °C. In all cases, the reactions were
stopped by transferring the agarose blocks into solution containing 0.2 M NaEDTA (pH 8.0), 1% SDS, and 1 mg/ml proteinase K.
Subsequent steps were carried out as described above for experiments
with topoisomerase II-mediated DNA cleavage.
Enzyme Treatment of Naked DNA in Agarose
Blocks
Cells embedded in agarose were permeabilized and
deproteinized as described above. The agarose blocks were washed four
times for 1 h each at 37 °C in 10 mM Tris-HCl (pH 7.5), 1
mM NaEDTA. The blocks containing genomic DNA were then
incubated for 1 h at 0 °C in NotI digestion buffer (New
England Biolabs Inc.) or, for induction of DNA cleavages by S1 nuclease
on the partially denaturated DNA, in 30 mM sodium acetate (pH
5.0), 50 mM NaCl, 2 mM ZnCl, and 50%
formamide. The blocks were then transferred to the appropriate buffer
containing the relevant enzyme. Treatment with NotI (40
units/sample; New England Biolabs Inc.) was carried out for 8 h at 37
°C, and reactions were terminated by the addition of NaEDTA to a
concentration of 20 mM. Treatment with S1 nuclease (Sigma) was
carried out for 1 h at 25 °C and was terminated as described above.
Pulsed Field Gel Electrophoresis (PFGE), Southern
Transfer, and Hybridization
Pieces of agarose blocks containing
DNA from 10
cells were placed into the wells of 1%
agarose gels cast in TAE buffer (40 mM Tris acetate (pH 8.0),
2 mM NaEDTA), and PFGE was carried out in the Bio-Rad Mapper
system in TAE buffer at 13 °C for 28 h at a voltage gradient of 6
V/cm with the switch time ramped linearly from 20 to 90 s in the
two-state mode. Concatamers of the phage
DNA (Bio-Rad) were used
as markers. Gels were stained with ethidium bromide and photographed
under UV light. For Southern transfer, gels were incubated in 0.25 M HCl for 15 min at room temperature, washed twice in water
for 10 min, and incubated in 0.4 M NaOH for 30 min. Alkaline
transfer of DNA to Hybond N
membranes (Amersham Corp.)
was carried out according to the supplier's instructions.
Prehybridization for 5-10 h and hybridization for 12-16 h
were carried out in an Autoblot hybridization incubator (HYBAID) at 65
°C in the following solution: 5
SSPE (10 mM sodium
phosphate (pH 7.5), 180 mM NaCl, and 1 mM NaEDTA), 5
Denhardt's solution (0.02% (w/v) bovine serum albumin,
0.02% (w/v) Ficoll, and 0.02% polyvinylpyrrolidone), 20 mM NaEDTA (pH 8.0), 0.5% (w/v) SDS (Bio-Rad), and 400 µg/ml
denatured salmon sperm DNA. Radioactive probes were prepared using the
Megaprime labeling kit (Amersham Corp.). After hybridization, the
filters were washed for 1 h at 65 °C in 2
SSPE, 0.5% SDS;
for 1 h at 65 °C in 0.5
SSPE, 1% SDS; and for 1 h at 65
°C in 0.1
SSPE, 1% SDS. They were then exposed to Fuji RX
film at -75 °C with an intensifying screen (DuPont NEN).
Size Distribution of Genomic DNA Fragments Released
after Mild Digestion of Nuclear DNA with Endogenous and Exogenous
Nucleases
To analyze patterns of nuclear DNA cleavage by
exogenous nucleases, one has to keep in mind the possibility of
simultaneous DNA fragmentation by endogenous nucleases. Therefore, we
began with analyzing the pattern of DNA fragmentation by endogenous
enzymes. The best known endogenous nucleases are Ca-
and Mg
-dependent nucleases and DNase II(12) .
These enzymes have different pH optima and different requirements for
divalent cations. The Ca
- and
Mg
-dependent nucleases could be expected to be fully
active under the conditions we used for treatment of permeabilized
cells with exogenous DNase I and micrococcal nuclease, i.e. in
buffer A (see ``Materials and Methods'' and below). DNase II
has maximal activity at pH 5.0 and might therefore cause DNA
degradation under the conditions used for treatment of permeabilized
cells with mung bean and S1 nucleases (buffer B). The activity of DNase
II can, however, be discriminated due to the ability of the enzyme to
digest DNA in the absence of divalent cations (buffer C). Based on
these considerations, we studied the kinetics of nuclear DNA cleavage
by endogenous nucleases in three different buffers: buffers A-C.
to buffer A (Fig. 1A, rightpanel). This result
demonstrates that at neutral pH, degradation of DNA in permeabilized
cells is accomplished by endogenous Ca
- and
Mg
-dependent nucleases, which are known to be
inhibited by Zn
(12) .
and
Mg
(buffer A) (leftpanel) or with
Ca
, Mg
, and 2 mM
Zn
(rightpanel). The time of
incubation at 37 °C is indicated above the lanes. B,
incubation of permeabilized cells in acidic buffer (pH 5.0)
supplemented with Zn
(buffer B) (leftpanel) or with NaEDTA (buffer C) (rightpanel). The time of incubation at 37 °C is indicated
above the lanes.
DNA concatamers within the size range of 50-500 kb
under their electrophoretic conditions, as we did in our experiments (cf.Fig. 2C, lane13), thus
achieving the linear distribution of DNA fragments within this size
range. It is also worth mentioning that other authors, who used PFGE
for separation of large apoptotic DNA fragments, have not observed any
discontinuity in the size distribution of the DNA
fragments(14) .
DNA concatamers) are shown to
the right of lane6. B, cells were treated
for 30 min at 0 °C with increasing concentrations of DNase I. Lanes 2-7 represent DNA from cells treated with 2, 5,
10, 25, 50, and 80 mg of DNase I, respectively, in 1 ml of buffer A. Lane1 represents DNA from cells incubated in buffer
A without enzyme. C, cells were treated for 30 min at 0 °C
with increasing concentrations of micrococcal nuclease (MNase). Lanes2-10 represent DNA from
cells treated with 0.005, 0.01, 0.02, 0.04, 0.1, 0.2, 0.4, 0.8, and 1.6
units of micrococcal nuclease, respectively, in 1 ml of buffer A. Lane1 represents DNA from cells incubated in buffer
A without enzyme. Lanes11 and 13 are
autoradiograms of
DNA and phage
DNA concatamers,
respectively. Lane12 represents the autoradiogram of
the same DNA as separated in lane10. A mixture of
total DNA from OC-NYH-VM cells and of phage
DNA, labeled by nick
translation, was used as a hybridization probe. D, shown is
the separation of micrococcal nuclease-mediated DNA cleavage products
under conditions permitting resolution in the 5-75-kb size
region. Lanes 2-4 represent DNA from permeabilized cells
treated with 0.4, 0.8, and 1.6 units of micrococcal nuclease,
respectively. Lane1 shows the distribution of a 5-kb
ladder size marker (M) (Bio-Rad).
and Mg
(Fig. 1A). Hence, these conditions were used for
treatment of permeabilized cells with exogenous nucleases. The
experiments were carried out as described above, except that the time
of incubation was kept constant, while the concentration of exogenous
enzymes was varied. Analysis of the size distribution of large
fragments of genomic DNA released as a result of treatment of
permeabilized cells with mung bean nuclease, S1 nuclease, and DNase I (Fig. 2, A and B) did not reveal an
accumulation of any discrete intermediate digestion products. On the
contrary, treatment of permeabilized cells with micrococcal nuclease
caused rapid accumulation of 50-kb DNA fragments (Fig. 2C), which were similar to those described by
other researchers(2, 3) . We noticed, however, that
the distribution of DNA cleavage products separated by PFGE under
conditions adequate for resolution in the 50-kb to 1-megabase size
range showed a rather lower limit below 50 kb (Fig. 2, A-C). Furthermore, in an experiment with micrococcal
nuclease digestion (Fig. 2C), a relatively sharp band
in the 50-kb region was seen only after extensive digestion, when most
of the high molecular mass fragments had disappeared from the gel. This
indicates that the 50-kb band might be an artifact of the separation
procedure, and indeed, separation of the same samples under conditions
favorable for resolution of fragments in the size range of 5-75
kb led to an estimate for the size of the accumulated fragments of
25-30 kb (Fig. 2D). However, the apparent band
was located exactly in the region where the linear correlation between
the electrophoretic mobility and molecular mass is replaced by a
logarithmic correlation. Hence, the apparent concentration of the
material in the 30-kb region might also be the result of compression in
this particular area under the conditions that have been used in this
set of experiments.
Analysis of the Cleavage Specificity of Different
Nucleases in an Amplified Human c-myc Gene Locus
To study the
specificity of long-range fragmentation of eukaryotic DNA by different
nucleases, we combined the separation of released DNA fragments by PFGE
with an indirect end labeling of cleavage products bearing the third
exon of the c-myc gene. In all experiments, the DNA samples
were treated with NotI endonuclease before separation by PFGE.
The distribution of NotI sites within the amplified c-myc gene locus in OC-NYH-VM cells has been characterized previously (7, 8) and is shown in Fig. 3A. The
third exon of the c-myc gene maps to the left end of an
800-kb NotI fragment starting within the c-myc gene and extending downstream, which is clearly seen on Southern
blots hybridized with the c-myc probe (Fig. 3-5).
This is the only fragment seen after digestion of control DNA samples
with NotI restriction endonuclease (Fig. 3, B, lane8; and C, lane6);
the high molecular mass band with an apparent size of 1 megabase is a
compression artifact of the separation method as it is also seen in
the marker lane (Fig. 3, B, lane 1; and C, lane 1).
(buffer B (lanes4 and 5)). After separation by PFGE and Southern
transfer, fragments bearing the third exon of the c-myc gene
were visualized by hybridization. Lane1 shows the
distribution of phage
DNA concatamers. Lane6 demonstrates the distribution of the DNA products of topoisomerase
II-mediated cleavage in vivo.Lane7 demonstrates the distribution of the DNA products of topoisomerase
II-mediated cleavage generated after extraction of nuclei with 2 M NaCl. Lane8 represents the control DNA sample
from OC-NYH-VM cells digested with the NotI restriction
enzyme. C, permeabilized cells were treated for 10 min at 37
°C in 1 ml of buffer B with 300, 150, and 50 units of S1 nuclease (lanes 2-4), respectively, or with 60, 150, and 300
units of mung bean nuclease (lanes 8-10), respectively.
After separation by PFGE and Southern transfer, fragments bearing the
third exon of the c-myc gene were visualized by hybridization. Lanes6 and 7 represent DNA from untreated
cells (control (C)) (lane6) and from cells
incubated in buffer B without enzymes (lane7). Lane5 demonstrates the distribution of DNA products
of topoisomerase II-mediated cleavage in vivo. The
distribution of phage
DNA concatamers is shown in lane1. M, marker DNA.
or
Ca
as a similar specific pattern of DNA fragmentation
was observed in buffer C containing 2 mM EDTA (Fig. 3B, lanes2 and 3).
Furthermore, in contrast to neutral Ca
- and
Mg
-dependent nucleases (Fig. 1A),
endogenous acidic nucleases were not inhibited by Zn
present in buffer B (Fig. 3B, lanes 4 and 5). Taken together, these observations suggest that
an endogenous DNase II or a closely related enzyme is responsible for
digesting nuclear DNA at pH 5.0.
- and
Mg
-dependent nucleases (Fig. 4A).
However, the hybridization background was markedly higher, indicating
that cleavages also occur at other positions. Nevertheless, it is
obvious that the cleavage pattern generated by the endogenous neutral
Ca
- and Mg
-dependent nucleases is
not random because of the more or less regularly spaced bands seen in
the size range of 300 kb to 1 megabase. However, the nature of this
regular pattern is not presently clear.
- and Mg
-dependent nucleases (A), DNase I (B), and micrococcal nuclease (C). A, permeabilized cells were incubated for 20,
40, 60, 80, and 100 min (lanes 2-6, respectively) at 37
°C in neutral buffer supplemented with Ca
and
Mg
(buffer A). After separation by PFGE and Southern
transfer, fragments bearing the third exon of the c-myc gene
were visualized by hybridization. Lane1 demonstrates
the distribution of the DNA products of topoisomerase II-mediated
cleavage in vivo. B, lanes 1-4 represent DNA
samples from permeabilized cells treated with 0, 10, 25, and 50 µg
of DNase I, respectively, in 1 ml of buffer A. After separation by PFGE
and Southern transfer, fragments bearing the third exon of the
c-myc gene were visualized by hybridization. Lane6 demonstrates the distribution of DNA products of
topoisomerase II-mediated cleavage in vivo. The distribution
of phage
DNA concatamers is shown in lane5. M,
marker DNA. C, lanes 2-8 represent DNA from
permeabilized cells treated with 0.01, 0.04, 0.1, 0.2, 0.4, 0.8, and
1.6 units of micrococcal nuclease (MNase), respectively, in 1
ml of buffer A. After separation by PFGE and Southern transfer,
fragments bearing the third exon of the c-myc gene were
visualized by hybridization. Lane1 demonstrates the
distribution of DNA products of topoisomerase II-mediated cleavage in vivo.
- and Mg
-dependent
nucleases, we studied the specificity of cleavages of the same genomic
area by two exogenous nucleases active at neutral pH, namely DNase I
and micrococcal nuclease. Permeabilized cells were incubated for 30 min
in buffer A with increasing concentrations of exogenous nuclease. To
diminish the activity of endogenous nucleases, incubations were carried
out at 0 °C. DNase I generated a pattern of large DNA fragments (Fig. 4B) that was essentially similar to patterns
generated by endogenous acidic nucleases, single-stranded DNA-specific
nucleases, and nuclear matrix-bound topoisomerase II. More important,
this specific pattern of long-range cleavage of the c-myc amplicon by DNase I was not observed when the high salt-extracted
nuclei were treated with the same enzyme (Fig. 5A).
Hence, the specificity of DNase I-mediated long-range fragmentation of
the c-myc amplicon is defined at the level of DNA packaging in
chromatin. Extraction of histones and some non-histone proteins
disrupts the chromatin organization and abolishes the specificity of
DNase I-mediated cleavage.
Fragmentation of Naked DNA by S1 Nuclease
The
preferential sensitivity of matrix-attached DNA to S1 and mung bean
nucleases indicates that these DNA regions might possess a noncanonical
secondary structure. In the simplest case, the distribution of
preferential targets for single strand-specific nucleases could be
determined by the distribution of easily melting sequences along the
DNA helix. It has indeed been demonstrated that the ability to melt
under relatively mild conditions constitutes an intrinsic property of
matrix-associated region elements, DNA sequences that have specific
affinity for some binding sites in the isolated nuclear
matrix(17) . Furthermore, easily melting DNA sequences have
been reported to partition eukaryotic DNA into domains similar in size
to DNA loops fixed at the nuclear matrix (18) . Hence, it
seemed reasonable to examine whether the specific pattern of
fragmentation of the c-myc amplicon by single strand-specific
nucleases may be determined by the distribution of low-melting DNA
sequences. With this aim, we prepared agarose blocks containing naked
DNA from OC-NYH-VM cells, treated them with S1 nuclease under
conditions favoring local denaturation of easily melting DNA
sequences(18) , and then carried out the Southern analysis
described above. No specific fragmentation of the partially denatured
DNA by S1 nuclease was observed in this experiment (Fig. 5B). Hence, it seems likely that preferential S1
targets in permeabilized cells are created by interaction of DNA with
nuclear matrix components.
-globin genes(15) . The modification
of DNA double-helix structure probably originates as a result of
interaction of DNA with nuclear matrix components because the same
regions do not constitute preferential targets for S1 cleavage in
partially denatured naked DNA. It is important to emphasize that
matrix-attached DNA is preferentially sensitive to nucleases only when
organization of DNA in chromatin is preserved. Extraction of histone
with 2 M NaCl solution abolishes this preferential sensitivity (Fig. 5A). At the same time, the matrix attachment
regions can be preferentially cleaved by topoisomerase II both in
living cells and in high salt-extracted nuclei (Fig. 3B, lanes 6 and 7). Thus, the
specificity of cleavage of the matrix-attached DNA by endogenous
topoisomerase II could not be explained simply by preferential
accessibility of this DNA in chromatin. It is more likely that
matrix-bound DNA permanently interacts with topoisomerase II (and
another matrix-associated proteins). This interaction may disturb
normal chromatin organization, resulting in preferential accessibility
of matrix attachment areas. Another explanation of the preferential
accessibility of matrix-attached DNA is based on a particular model of
the nuclear matrix organization that has been discussed previously (22) .
We are indebted to Dr. Ole Westergaard for many
valuable discussions. We also thank Dr. Ronald Hancock for helpful
discussions and critical comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.