©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Long-range Fragmentation of the Eukaryotic Genome by Exogenous and Endogenous Nucleases Proceeds in a Specific Fashion via Preferential DNA Cleavage at Matrix Attachment Sites (*)

(Received for publication, March 3, 1995; and in revised form, June 6, 1995)

Irina I. Gromova (1) (2)(§) Ole F. Nielsen (1) Sergey V. Razin (2)

From the  (1)Department of Molecular Biology, University of Aarhus, C. F. Mollers Alle 130, 8000 Aarhus C, Denmark and the (2)Institute of Gene Biology, Russian Academy of Sciences, Vavilov Street 34/5, 117334 Moscow, Russia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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- and Mg-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.


INTRODUCTION

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(^1)(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.

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.


MATERIALS AND METHODS

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 10^7 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 10^6 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-beta-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(2) (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(2), and 1.5 mM CaCl(2)) 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(2)), 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(2) 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(2), 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 10^6 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).


RESULTS

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.

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 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) .


Figure 1: Fragments of genomic DNA released by endogenous nucleases. A, incubation of permeabilized cells in neutral buffer supplemented with Ca 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.



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 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) .


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 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).



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 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).


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 (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.



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 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.

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- 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.


Figure 4: Mapping of DNA cleavages downstream from the c-myc gene generated by endogenous neutral Ca- 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.



Having characterized the pattern of cleavages within the c-myc amplicon by endogenous neutral Ca- 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.


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.

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.


DISCUSSION

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 alpha-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) .

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.


FOOTNOTES

*
This work was supported by grants from the Danish Center for Human Genome Research, the Danish Cancer Society, the Astrid Thaysen Foundation, and the Novo Foundation (to Ole Westergaard). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, University of Aarhus, C. F. Mollers Alle 130, 8000 Aarhus C, Denmark.

^1
The abbreviations used are: kb, kilobase(s); PFGE, pulsed field gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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