Nucleic Acids Research, Vol. 20, No. 3 467-473
The recognition of DNA cleavage sites topoisomerase 11
by
porcine spleen
Hurng-Wern Huang, Jin-Kai Juang and Hon-Ju Liu* Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China Received October 28, 1991; Revised and Accepted January 14, 1992
ABSTRACT The cutting sites specificity of topoisomerase 11 from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase 11 prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase 11 seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase 11 has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base. INTRODUCTION DNA topoisomerase II is a nuclear enzyme manipulating the topological structure of DNA(1). Strong evidences have revealed that topoisomerase II is necessary for many biological functions such as DNA replication and recombination(2 -4), Some reports also indicated that topoisomerase II may be involved in hsp7O gene expression(5). In addition its enzymatic functions, topoisomerase II also acts as a major component of chromsomal scaffold(6 -8). Despite being an important enzyme in cell nucleus, its enzymatic mechanism is still obscure and remains merely as models preposed by J.C. Wang(9), L.F. Liu(IO), and recently by Eamshaw(1 1). In Liu's model, topoisomerase II binds to DNA, thereby cuts the double strand and let another region of the DNA double strand passing through the break, then close the gap by ligation to make another circular topological isoform DNA. This model is supported by the fact that a certain kind of antitumor drugs, such as mAMSA(12) and VM26, can stablize a covalent complex of DNA-topoisomerase II which can be digested by proteinase K to reveal that there should have happened the cutting of single strand following with the cutting of complementary strand DNA, that can be demonstrated by gel electrophoresis. The DNA cleavage, in case of Drosophila topoisomerase 11(14), was reported to cut at the site of 3' end of pyrimidine. Some interesting cases indicated the site cutting specificity was also affected by the kind of anti-tumor drugs supplemented. For instance, Spitzner et al. demonstrated that chicken topoisomerase
*
To whom
correspondence
should be addressed
II usually localizes at the 3' side of pyrimidine base with the drugs effect of mAMSA, or preferably cleaves alternatively the purinepyrimidine repeats region of DNA with the effect of VM-26 (15). Capranico et al. (16) found that recognition of 3'-A site is required for the doxorubicin-stablized cleavage by topoisomerase II from mouse leukemia L1210 cells. In addition, it was observed by Andersen et al. (17) that the DNA cleavage by Drosophila topoisomerase II are not equal in term of cutting frequencies for its two complementary DNA strands under certain reaction condition. It suggested that the approach of topoisomerase II to the DNA double strands may be asymmetric by strand preference to select a specific strand of DNA for the first cutting. It is interesting in understanding the cutting pattern of porcine topoisomerase II and try to know how it does to recognize DNA toward its cutting site.
MATERIALS AND METHODS Materials VM-26 is a gift from Huang, J.-L. at IMB, Academic Sinica, ROC. Plasmid pX-3, a derivative of pH2.3(18) containing human heat shock gene hsp7O, is from Lai, Y.-C. at our institute. Enzymes were purchased from Boehringer Mannheim. ,y-[32P]-ATP and a-[35S]-ATP were purchased from NEN. T7 DNA sequencing kit was purchased from Promega and the other chemicals were from Merck or Sigma. Plasmid DNA was isolated by alkaline method(19) and purified further by centrifugating with two steps CsCl density gradient(20). Positive supercoiled DNA was prepared according to our previous method (21). Porcine DNA topoisomerase II was isolated as described by Juang et al. (22). Porcine spleen MAR(Matrix Association Region) DNA was isolated as described by Gasser et al. ( 23) . The isolated MAR was inserted into the Sma I site of plasmid pUC19. After transformation into E. coli DH5a competent cells, 36 clones were randomly selected (Al -A21, the white clones; BI -B9, the slightly blue ones and C1 -C6, the dark blue ones on IPTG-Xgal plate). The MARs DNA sequences were determined by T7 DNA polymerase sequencing system.
468 Nucleic Acids Research, Vol. 20, No. 3 Assay of Topoisomerase II DNA relaxation assay was performed in standard reaction buffer(5OmM Tris-HCl, pH7.5, lOOmM KCI, 5mM MgCl2, O.1mM EDTA, lmM ATP, 0.5mM DTT and 30Itg/ml BSA) with topoisomerase 11 and DNA sample at 37°C for 1 hour. Construction of Vectors The 2.7 kb fragment of pX-3 DNA (see MATERIALS) was digested by Pst I and inserted into the same site in M13 phage mpl 1. The recombinant DNA was further subcloned after suitabily digested with T4 DNA polymerase as described by Dale and Arrow(24). From this a nest of 16 clones were selelcted as the subclones from number 1 to number 16. Then, the single strand phage DNA was prepared for sequence analysis.
Determination of Porcine Topoisomerase II Cutting Site Fragmentation of DNA by topoisomerase II-VM26 induced DNA damage at its cutting sites provides us the method to determine the cutting end by sequencing the DNA fragment. Two methods are used, one is developed by Hsieh, T.-s. (28) and the other is developed by us. Our method is briefly described as the following, S reactions which include 4 (G, A, T and C) reactions and 1 topoisomerase 11 cutting site assay reaction were performed side by side simultaneously. Becuase that the fragment made by Topoisomerase II-VM-26 breakage should have the same length as the fragment terminated at dideoxynucleotide by sequencing method, we can directly find out the topoisomerase II cutting site on the sequencing gel. We chose the DNA sequencing method developed by Tabor and Richardson(25). 2 tg DNA template and primer (1:1 molar ratio to template) were mixed in T7 DNA polymerase reaction buffer(40 mM Tris-HCl pH7.5, 10 mM MgCl2, 50 mM NaCl and 0.02 mM DTT) before incubated at 37°C for 15 min. The labeling reaction was performed by adding the following reagents: lul of 100 mM DTT, 2 yd of labeling mix (dGTP, dCTP, dTTP, each 1.5 ,uM), 1I1 of [a-35S]dATP (10 mCi/ml,with specific acitivity of 500 Ci/mmol on the arrival date and used within one month) and 3 units T7 DNA polymerase. Then the mixture was incubated for 5 min at room temperature. 5 tubes each labeled with (G), (A), (T), (C), (Topo) respectively were prepared. For the first four (G), (A), (T), (C) tubes, 2.5 jd of corresponding dideoxy nucleotide mixture (i.e., for G reaction, 81sM ddGTP & 85 itM each of four deoxynucleotides; for A reaction, 6 ytM ddATP & 85 ,M each of four deoxynucleotides; for T reaction, 9 ytM ddTTP & 85 tM each of four deoxynucleotides and for C reaction, 8 ,gM ddCTP & 85 yM each of four deoxynucleotides) was added to each tube, and for the (Topo) tubes, 2.5 yd of nucleotide mixture(dGTP, dATP, dTTP, dCTP, each 100 ,tM) was added. After the labeling reaction, added 3.5 11 labeling mixture to each of the (G), (A), (T), (C) and (Topo) tubes, and incubated them for 5 min at 37°C. The reactions in (G), (A), (T), (C) tubes were stopped by adding 4 tul of stop soliution (95 % formamide, 0.01% bromophenol blue and 0.01% xylene cyanole). The reaction mixture of the tubes labelled with (Topo) was used to perform the topoisomerase II cutting site assay. Topoisomerase II cutting site assay buffer was same as the standard topoisomerase 11 relaxation assay buffer except that VM-26 (which is used to stablilize the topoisomerase H-DNA complex) was added to final concentration of 20 tiM. After incubation at 37°C for 15 min, the cleavable complex was digested with lmg/ml of proteinase
K with the presence of 0.5% SDS and 10 mM EDTA at 50°C for 30 min. And then the reaction was stopped by adding 5 Al stop solution. In Hsieh's method, 32P end-labelled DNA was used as the substrate of topoisomerase I. A topoisomerase 11 cutting site reaction was performed by mixing topoisomerase II with endlabelled DNA in topoisomerase II cutting site assay buffer. The reaction was incubated at 37°C for 15 min then stopped with 0.5% SDS-IOmM EDTA solution. On the other hand, the chemical sequencing reaction was performed with the same endlabelled DNA as substrate, then the results of reactions were shown on sequencing gel to determine the location of topoisomerase II cutting site.
Estimation of the Half Turn Internal Energy of DNA Backbone The half turn internal energy(HTE) at a certain base pair (numbered as 'i') in a DNA backbone was estimated as the sum of internal energy formation for base pairing and base stacking in a string of DNA(26). The length of DNA under consideration is the section from the base pair at the number 'i -2' to the number 'i + 2' (thus 5 base pairs, two base pairs before the number 'i' and two base pairs after the number 'i' plus the one base pair 'i', were taken as one section). For each DNA string of this five base pairs, we consider five sets of hydrogen bonds formed by their 5 base pairing and four sets of bases stacking between each neighboring pairs located within 'i -2' to 'i +2' base pairs section. The boundary effect outside this section was neglected in our estimation.
RESULTS Topoisomerase II preparation without topoisomerase I and other endonuclease acitivity Since we look for the specific cutting site of topoisomerase H by sequencing DNA from topoisomerase 11-drug mediated DNA damage reaction. The purity of topoisomerase II prepartion without endonuclease or topoisomerase I contamination which
Figure 1. DNA relaxation activity of topoisomerase II. Positive superhelical
pUCl9 DNA (lane 1) was prepared from natural form I DNA and relaxed (lane 2) by topoisomerase II as described in Materials and Methods. Four kinds of DNA topoisomer (Lk=n, n-1, n-2 and n-3) each of single kind Lk were isolated from positive superhelical DNA collection in lane 2 by electro-elution, then each of them was either loaded directly to the gel without enzyme treatment (lane 3 for Lk of n-3; lane 4 for Lk of n-2; lane 5 for Lk of n-I and lane 6 for Lk of n), or relaxed by porcine DNA topoisomerase II preparation in the absence of ATP for electrophoresis (lane 7, 8, 9, and 10 for the DNA with Lk of n-3, n -2, n-I and n as substrate respectively); 15 min (lane I 1, 12, 13, and 14 for Lk of n-3, n-2, n- 1 and n respectively) or 30 min (lane 15, 16, 17 and 18 for Lk of n-3, n-2, n- I and n respectively).
Nucleic Acids Research, Vol. 20, No. 3 469 might cause non-topoisomerase II specific cutting of DNA is critically important. This possiblity is excluded by the experiment result shown in Fig. 1 where the topoisomerase H acitivity was demonstrated without detectable contamination of the other nuclease activity. That is to say, the relaxation of DNA by linking number was changed only by even number steps and no detectable band was seen changed by one stepwise or random stepwise migration in the gel. Determination of the Porcine Spleen Topoisomerase II DNA Cutting Site mAMSA, a kind of DNA intercalating type topoisomerase II inhibitor, could promote more DNA damage on a negative superhelical DNA than on a positive one (our unpublished data). Another topoisomerase II inhibitor, VM26, is believed to bind into the minor groove site of DNA helix (27). We had checked previously that the extent of topoisomerase 11-DNA damage caused by VM26 did not change with the negative or positive superhelicity of DNA. So, VM26 was selected in the cleavage experiment with topoisomerase II. The DNA fragments used to study the topoisomerase H cutting sequences were a 2.7 kb Pst I cut fragment from plasmid pX-3 and a MAR vectors constructed by us. The pX-3 fragment contains 3' end of Apr gene, ori site of pAT 153 and 5' control region of human hsp70 gene. It bound to porcine topoisomerase II strongly as that was revealed by gel retardation assay (data not shown). The DNA cutting site was determined by dideoxy method or chemical method. Both methods gave us the same result. Because the 2.7 kb Pst fragment was too large for sequencing, so we subcloned their sub- fragments into a nest of 16 clones which are assigned as the subclone 1 to 16. An example of the results (for the subclone 3) using dideoxy method is shown in Fig. 2. From the 16 subclones sequenced and subjected to topoisomerase II cutting site experiment, 12 strong (high frequency) cutting sites were found (Table I). We could not find a consensus sequence from these 12 topoisomerase DNA cutting sites by comparing 21 flanking bases of them (from -10 to + 10 bases, which refer to the upstream and downstream sequences
respectively). Nonetheless, we find that 9 sites among the 12 strong cutting sites were of 'A' base at 3' end rather than 'C' or 'T' as that had been reported by Sander and Hsieh(28). The denatured pUC 19 superhelix DNA to which a MAR of 135 base pairs from porcine spleen had been inserted into its Sma I site
(D
The recognition of DNA cleavage sites topoisomerase 11
by
porcine spleen
Hurng-Wern Huang, Jin-Kai Juang and Hon-Ju Liu* Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China Received October 28, 1991; Revised and Accepted January 14, 1992
ABSTRACT The cutting sites specificity of topoisomerase 11 from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase 11 prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase 11 seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase 11 has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base. INTRODUCTION DNA topoisomerase II is a nuclear enzyme manipulating the topological structure of DNA(1). Strong evidences have revealed that topoisomerase II is necessary for many biological functions such as DNA replication and recombination(2 -4), Some reports also indicated that topoisomerase II may be involved in hsp7O gene expression(5). In addition its enzymatic functions, topoisomerase II also acts as a major component of chromsomal scaffold(6 -8). Despite being an important enzyme in cell nucleus, its enzymatic mechanism is still obscure and remains merely as models preposed by J.C. Wang(9), L.F. Liu(IO), and recently by Eamshaw(1 1). In Liu's model, topoisomerase II binds to DNA, thereby cuts the double strand and let another region of the DNA double strand passing through the break, then close the gap by ligation to make another circular topological isoform DNA. This model is supported by the fact that a certain kind of antitumor drugs, such as mAMSA(12) and VM26, can stablize a covalent complex of DNA-topoisomerase II which can be digested by proteinase K to reveal that there should have happened the cutting of single strand following with the cutting of complementary strand DNA, that can be demonstrated by gel electrophoresis. The DNA cleavage, in case of Drosophila topoisomerase 11(14), was reported to cut at the site of 3' end of pyrimidine. Some interesting cases indicated the site cutting specificity was also affected by the kind of anti-tumor drugs supplemented. For instance, Spitzner et al. demonstrated that chicken topoisomerase
*
To whom
correspondence
should be addressed
II usually localizes at the 3' side of pyrimidine base with the drugs effect of mAMSA, or preferably cleaves alternatively the purinepyrimidine repeats region of DNA with the effect of VM-26 (15). Capranico et al. (16) found that recognition of 3'-A site is required for the doxorubicin-stablized cleavage by topoisomerase II from mouse leukemia L1210 cells. In addition, it was observed by Andersen et al. (17) that the DNA cleavage by Drosophila topoisomerase II are not equal in term of cutting frequencies for its two complementary DNA strands under certain reaction condition. It suggested that the approach of topoisomerase II to the DNA double strands may be asymmetric by strand preference to select a specific strand of DNA for the first cutting. It is interesting in understanding the cutting pattern of porcine topoisomerase II and try to know how it does to recognize DNA toward its cutting site.
MATERIALS AND METHODS Materials VM-26 is a gift from Huang, J.-L. at IMB, Academic Sinica, ROC. Plasmid pX-3, a derivative of pH2.3(18) containing human heat shock gene hsp7O, is from Lai, Y.-C. at our institute. Enzymes were purchased from Boehringer Mannheim. ,y-[32P]-ATP and a-[35S]-ATP were purchased from NEN. T7 DNA sequencing kit was purchased from Promega and the other chemicals were from Merck or Sigma. Plasmid DNA was isolated by alkaline method(19) and purified further by centrifugating with two steps CsCl density gradient(20). Positive supercoiled DNA was prepared according to our previous method (21). Porcine DNA topoisomerase II was isolated as described by Juang et al. (22). Porcine spleen MAR(Matrix Association Region) DNA was isolated as described by Gasser et al. ( 23) . The isolated MAR was inserted into the Sma I site of plasmid pUC19. After transformation into E. coli DH5a competent cells, 36 clones were randomly selected (Al -A21, the white clones; BI -B9, the slightly blue ones and C1 -C6, the dark blue ones on IPTG-Xgal plate). The MARs DNA sequences were determined by T7 DNA polymerase sequencing system.
468 Nucleic Acids Research, Vol. 20, No. 3 Assay of Topoisomerase II DNA relaxation assay was performed in standard reaction buffer(5OmM Tris-HCl, pH7.5, lOOmM KCI, 5mM MgCl2, O.1mM EDTA, lmM ATP, 0.5mM DTT and 30Itg/ml BSA) with topoisomerase 11 and DNA sample at 37°C for 1 hour. Construction of Vectors The 2.7 kb fragment of pX-3 DNA (see MATERIALS) was digested by Pst I and inserted into the same site in M13 phage mpl 1. The recombinant DNA was further subcloned after suitabily digested with T4 DNA polymerase as described by Dale and Arrow(24). From this a nest of 16 clones were selelcted as the subclones from number 1 to number 16. Then, the single strand phage DNA was prepared for sequence analysis.
Determination of Porcine Topoisomerase II Cutting Site Fragmentation of DNA by topoisomerase II-VM26 induced DNA damage at its cutting sites provides us the method to determine the cutting end by sequencing the DNA fragment. Two methods are used, one is developed by Hsieh, T.-s. (28) and the other is developed by us. Our method is briefly described as the following, S reactions which include 4 (G, A, T and C) reactions and 1 topoisomerase 11 cutting site assay reaction were performed side by side simultaneously. Becuase that the fragment made by Topoisomerase II-VM-26 breakage should have the same length as the fragment terminated at dideoxynucleotide by sequencing method, we can directly find out the topoisomerase II cutting site on the sequencing gel. We chose the DNA sequencing method developed by Tabor and Richardson(25). 2 tg DNA template and primer (1:1 molar ratio to template) were mixed in T7 DNA polymerase reaction buffer(40 mM Tris-HCl pH7.5, 10 mM MgCl2, 50 mM NaCl and 0.02 mM DTT) before incubated at 37°C for 15 min. The labeling reaction was performed by adding the following reagents: lul of 100 mM DTT, 2 yd of labeling mix (dGTP, dCTP, dTTP, each 1.5 ,uM), 1I1 of [a-35S]dATP (10 mCi/ml,with specific acitivity of 500 Ci/mmol on the arrival date and used within one month) and 3 units T7 DNA polymerase. Then the mixture was incubated for 5 min at room temperature. 5 tubes each labeled with (G), (A), (T), (C), (Topo) respectively were prepared. For the first four (G), (A), (T), (C) tubes, 2.5 jd of corresponding dideoxy nucleotide mixture (i.e., for G reaction, 81sM ddGTP & 85 itM each of four deoxynucleotides; for A reaction, 6 ytM ddATP & 85 ,M each of four deoxynucleotides; for T reaction, 9 ytM ddTTP & 85 tM each of four deoxynucleotides and for C reaction, 8 ,gM ddCTP & 85 yM each of four deoxynucleotides) was added to each tube, and for the (Topo) tubes, 2.5 yd of nucleotide mixture(dGTP, dATP, dTTP, dCTP, each 100 ,tM) was added. After the labeling reaction, added 3.5 11 labeling mixture to each of the (G), (A), (T), (C) and (Topo) tubes, and incubated them for 5 min at 37°C. The reactions in (G), (A), (T), (C) tubes were stopped by adding 4 tul of stop soliution (95 % formamide, 0.01% bromophenol blue and 0.01% xylene cyanole). The reaction mixture of the tubes labelled with (Topo) was used to perform the topoisomerase II cutting site assay. Topoisomerase II cutting site assay buffer was same as the standard topoisomerase 11 relaxation assay buffer except that VM-26 (which is used to stablilize the topoisomerase H-DNA complex) was added to final concentration of 20 tiM. After incubation at 37°C for 15 min, the cleavable complex was digested with lmg/ml of proteinase
K with the presence of 0.5% SDS and 10 mM EDTA at 50°C for 30 min. And then the reaction was stopped by adding 5 Al stop solution. In Hsieh's method, 32P end-labelled DNA was used as the substrate of topoisomerase I. A topoisomerase 11 cutting site reaction was performed by mixing topoisomerase II with endlabelled DNA in topoisomerase II cutting site assay buffer. The reaction was incubated at 37°C for 15 min then stopped with 0.5% SDS-IOmM EDTA solution. On the other hand, the chemical sequencing reaction was performed with the same endlabelled DNA as substrate, then the results of reactions were shown on sequencing gel to determine the location of topoisomerase II cutting site.
Estimation of the Half Turn Internal Energy of DNA Backbone The half turn internal energy(HTE) at a certain base pair (numbered as 'i') in a DNA backbone was estimated as the sum of internal energy formation for base pairing and base stacking in a string of DNA(26). The length of DNA under consideration is the section from the base pair at the number 'i -2' to the number 'i + 2' (thus 5 base pairs, two base pairs before the number 'i' and two base pairs after the number 'i' plus the one base pair 'i', were taken as one section). For each DNA string of this five base pairs, we consider five sets of hydrogen bonds formed by their 5 base pairing and four sets of bases stacking between each neighboring pairs located within 'i -2' to 'i +2' base pairs section. The boundary effect outside this section was neglected in our estimation.
RESULTS Topoisomerase II preparation without topoisomerase I and other endonuclease acitivity Since we look for the specific cutting site of topoisomerase H by sequencing DNA from topoisomerase 11-drug mediated DNA damage reaction. The purity of topoisomerase II prepartion without endonuclease or topoisomerase I contamination which
Figure 1. DNA relaxation activity of topoisomerase II. Positive superhelical
pUCl9 DNA (lane 1) was prepared from natural form I DNA and relaxed (lane 2) by topoisomerase II as described in Materials and Methods. Four kinds of DNA topoisomer (Lk=n, n-1, n-2 and n-3) each of single kind Lk were isolated from positive superhelical DNA collection in lane 2 by electro-elution, then each of them was either loaded directly to the gel without enzyme treatment (lane 3 for Lk of n-3; lane 4 for Lk of n-2; lane 5 for Lk of n-I and lane 6 for Lk of n), or relaxed by porcine DNA topoisomerase II preparation in the absence of ATP for electrophoresis (lane 7, 8, 9, and 10 for the DNA with Lk of n-3, n -2, n-I and n as substrate respectively); 15 min (lane I 1, 12, 13, and 14 for Lk of n-3, n-2, n- 1 and n respectively) or 30 min (lane 15, 16, 17 and 18 for Lk of n-3, n-2, n- I and n respectively).
Nucleic Acids Research, Vol. 20, No. 3 469 might cause non-topoisomerase II specific cutting of DNA is critically important. This possiblity is excluded by the experiment result shown in Fig. 1 where the topoisomerase H acitivity was demonstrated without detectable contamination of the other nuclease activity. That is to say, the relaxation of DNA by linking number was changed only by even number steps and no detectable band was seen changed by one stepwise or random stepwise migration in the gel. Determination of the Porcine Spleen Topoisomerase II DNA Cutting Site mAMSA, a kind of DNA intercalating type topoisomerase II inhibitor, could promote more DNA damage on a negative superhelical DNA than on a positive one (our unpublished data). Another topoisomerase II inhibitor, VM26, is believed to bind into the minor groove site of DNA helix (27). We had checked previously that the extent of topoisomerase 11-DNA damage caused by VM26 did not change with the negative or positive superhelicity of DNA. So, VM26 was selected in the cleavage experiment with topoisomerase II. The DNA fragments used to study the topoisomerase H cutting sequences were a 2.7 kb Pst I cut fragment from plasmid pX-3 and a MAR vectors constructed by us. The pX-3 fragment contains 3' end of Apr gene, ori site of pAT 153 and 5' control region of human hsp70 gene. It bound to porcine topoisomerase II strongly as that was revealed by gel retardation assay (data not shown). The DNA cutting site was determined by dideoxy method or chemical method. Both methods gave us the same result. Because the 2.7 kb Pst fragment was too large for sequencing, so we subcloned their sub- fragments into a nest of 16 clones which are assigned as the subclone 1 to 16. An example of the results (for the subclone 3) using dideoxy method is shown in Fig. 2. From the 16 subclones sequenced and subjected to topoisomerase II cutting site experiment, 12 strong (high frequency) cutting sites were found (Table I). We could not find a consensus sequence from these 12 topoisomerase DNA cutting sites by comparing 21 flanking bases of them (from -10 to + 10 bases, which refer to the upstream and downstream sequences
respectively). Nonetheless, we find that 9 sites among the 12 strong cutting sites were of 'A' base at 3' end rather than 'C' or 'T' as that had been reported by Sander and Hsieh(28). The denatured pUC 19 superhelix DNA to which a MAR of 135 base pairs from porcine spleen had been inserted into its Sma I site
(D
