Positioning the 3'-DNA Terminus for Topoisomerase II-mediated Religation*

Amy M. WilstermannDagger § and Neil OsheroffDagger ||

From the Departments of Dagger  Biochemistry and  Medicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, January 10, 2001, and in revised form, March 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Despite the importance of the topoisomerase II DNA cleavage/rejoining cycle to genomic integrity, the mechanistic details of religation are poorly understood. Topoisomerase II utilizes covalent protein-DNA interactions to align the 5'-termini of cleaved DNA for religation. However, because the enzyme does not form covalent bonds with the 3'-DNA termini, the basis for the alignment of the 3'-ends is less clear. Three major possibilities exist. The 3'-termini may be positioned for religation (i) by base pairing to their complementary DNA strands, (ii) by base stacking to the adjacent residues, or (iii) by noncovalent interactions with topoisomerase II. To distinguish between these possibilities, the ability of human topoisomerase IIalpha to religate a series of oligonucleotides with altered base pairing or base stacking at their 3'-termini was determined. Substrates containing modifications that disrupted terminal base pairing or base stacking with-out affecting the 3'-terminal base were resealed at wild-type rates. In contrast, substrates that lacked the terminal base (or contained an altered base) displayed very low rates of religation. On the basis of these results, we propose that topoisomerase II positions the 3'-DNA termini for religation through noncovalent protein-DNA contacts.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The ability to ligate DNA chains is essential to all forms of life (1-3). This activity is indispensable for nearly every process that the genetic material undergoes in the cell, including replication, recombination, and repair.

Although not ligases in the "classic" sense, topoisomerases routinely join DNA chains as part of their fundamental cellular activities (4-6). To interconvert different topological states of DNA, type II topoisomerases pass a double helix through a transient double-stranded break that is generated in a separate nucleic acid segment (7-9). If these enzymes do not successfully rejoin the double-stranded break, a number of deleterious events can occur including chromosomal insertions, deletions, and translocations (8, 10). In extreme cases, the build up of topoisomerase II-mediated DNA breaks triggers cell death pathways (11).

Because of the sensitivity of cells to the accumulation of these DNA breaks, the religation activity of topoisomerase II has been exploited as a target for cancer chemotherapy (8, 12-15). Drugs such as etoposide, which is front-line therapy for a variety of human malignancies (15, 16), increase topoisomerase II-generated DNA breaks specifically by inhibiting the ability of the enzyme to rejoin cleaved DNA molecules (8, 14, 17).

Despite the importance of the DNA religation reaction of topoisomerase II to the critical cellular functions and chemotherapeutic potential of the enzyme, the mechanistic details of this process are not completely understood. In contrast to ligases, topoisomerase II carries out its religation reaction within a covalent enzyme-DNA complex, known as the cleavage complex (see Fig. 1; Refs. 7-9). This complex is formed during the cleavage event so that the enzyme can maintain the integrity of the genetic material throughout the strand passage process. The bonds that link topoisomerase II to the DNA are established between active site tyrosyl residues on each subunit of the homodimeric enzyme and the 5'-terminal phosphates of the newly cleaved DNA strands (18-20). Because the points of topoisomerase II-mediated scission on the two strands are staggered by four base pairs, each cleaved chain within the cleavage complex contains a four base single-stranded cohesive end at its 5'-terminus (see Fig. 1).

In most cases, ligases rely heavily on base pairing to correctly position the DNA termini for ligation (21, 22). However, the covalent attachment of topoisomerase II to the 5'-termini of its ligation substrate obviates the need for base pairing. In fact, human topoisomerase IIalpha is able to efficiently religate substrates that contain four consecutive abasic sites within the terminal cohesive ends (thus eliminating all base pairing) (23). On this basis, it was proposed that the type II enzyme correctly positions the 5'-DNA termini for religation through covalent protein-DNA interactions (23).

The role of base pairing in the positioning of the 3'-DNA termini for religation is less clear. In contrast to the 5'-ends, the 3'-terminal residues remain base paired to their complementary strand following cleavage (see Fig. 1 and Ref. 18). Moreover, type II topoisomerases do not form covalent attachments with the 3'-DNA ends (18). As a result, it is not obvious whether the 3'-termini are positioned for religation through base pairing. Alternatively, they may be aligned by base stacking interactions with adjacent residues or through specific noncovalent contacts with the enzyme.

If the DNA cleavage/religation cycle of eukaryotic type II topoisomerases is to be fully understood, it is critical to determine how the enzyme positions the DNA termini for resealing. This issue takes on further importance because it is believed that many anticancer drugs that act by inhibiting topoisomerase II-mediated DNA religation do so by displacing the 3'-DNA termini within the cleavage complex (24-26). Therefore, to address the mechanism by which the enzyme positions the 3'-DNA termini, the ability of human topoisomerase IIalpha to religate a series of substrates with altered base pairing or base stacking at their 3'-termini was determined. Results indicate that enzyme-mediated DNA religation requires the presence of a 3'-terminal base, but does not require either terminal base pairing or base stacking. These findings suggest that topoisomerase II positions the 3'-DNA termini for religation through noncovalent protein-DNA contacts.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Preparation of Oligonucleotides-- A 40-base oligonucleotide corresponding to residues 87-126 of pBR322 (18, 27) and its complementary oligonucleotide were prepared on an Applied Biosystems DNA synthesizer. The sequences of the top and bottom strands were 5'-TGAAATCTAACAATGdown-arrow CGCTCATCGTCATCCTCGGCACCGT-3' and 5'-ACGGTGCCGAGGATGACGATGdown-arrow AGCGCATTGTTAGATTTCA-3', respectively. A second 40-base single-stranded oligonucleotide corresponding to residues 1072-1111 of the MLL oncogene (28, 29) and its complementary oligonucleotide also were synthesized. The sequences of the top and bottom strands were 5'-GCCTGGGTGACAAAGCdown-arrow AAAACACTGTCTCCAAAAAAAATT-3' and 5'-AATTTTTTTTGGAGACAGTGdown-arrow TTTTGCTTTGTCACCCAGGC-3', respectively. Points of topoisomerase II-mediated cleavage in both sequences are denoted by arrows. Single-stranded oligonucleotides containing a tetrahydrofuran abasic site analog or a deoxyuridine residue were prepared in a similar manner utilizing corresponding phosphoramidites obtained from Glen Research Corp. (Sterling, VA). Single-stranded oligonucleotides were labeled with [32P]phosphate on their 5'-termini with T4 polynucleotide kinase, purified by electrophoresis in a 7 M urea, 14% polyacrylamide gel, visualized by UV shadowing, excised, and eluted using the Qiagen extraction protocol. Complementary oligonucleotides were annealed by incubating equimolar amounts of each at 70 °C for 10 min and cooling to 25 °C (29).

Topoisomerase II-mediated DNA Cleavage-- Human topoisomerase IIalpha was expressed and purified from Saccharomyces cerevisiae as described previously (29, 30). DNA cleavage was monitored as described previously (29). Reaction mixtures contained 100 nM oligonucleotide in 19 µl of cleavage buffer (10 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 100 mM KCl, and 2.5% glycerol) that contained 5 mM MgCl2. Cleavage was initiated by the addition of 1 µl of human topoisomerase IIalpha (225 nM final concentration). Reactions were incubated at 37 °C for 10 min (unless stated otherwise) and stopped by the addition of 2 µl of 10% SDS followed by 1.5 µl of 250 mM EDTA. DNA cleavage products were digested with proteinase K, precipitated with ethanol, and resolved by electrophoresis in 7 M urea, 14% polyacrylamide gels. Reaction products were visualized and quantified using a Molecular Dynamics PhosphorImager system. Levels of DNA cleavage were calculated relative to that obtained with the wild-type substrate.

Topoisomerase II-mediated DNA Religation-- DNA religation assays were carried out using a modification of the procedure of Kingma and Osheroff (23). Cleavage/religation equilibria were established as described above in cleavage buffer containing 5 mM CaCl2. Kinetically competent topoisomerase II-DNA cleavage complexes were trapped by the addition of EDTA (final concentration, 6 mM). NaCl was added (final concentration, 500 mM) to prevent recleavage. Religation was initiated by the addition of MgCl2 (final concentration, 0.2 mM) and terminated by the addition of 2 µl of 10% SDS at times up to 60 s. Alternatively, DNA cleavage/religation equilibria were established as described in the previous section in cleavage buffer containing 5 mM MgCl2, and religation was initiated by transferring samples to 0 °C. Reactions were terminated as above. Samples were prepared for electrophoresis and analyzed as described above. The rate of DNA re-ligation was determined by quantifying the loss of cleaved DNA.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Given the importance of topoisomerase II-generated DNA breaks to the physiological and pharmacological functions of the enzyme, it is critical to understand the mechanism by which topoisomerase II reseals these breaks. During its scission event, the enzyme forms covalent bonds with the newly generated 5'-DNA termini (18-20). Topoisomerase II utilizes these covalent interactions to align the 5'-termini of the breaks for religation (Fig. 1) (23).


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Fig. 1.   Model for positioning the DNA termini for topoisomerase II-mediated religation. The 5'-DNA termini are properly positioned for religation within the topoisomerase II cleavage complex by covalent interactions with the enzyme rather than by base pairing between the single-stranded cohesive ends (23) (shown at left). The mechanism by which the 3'-DNA termini are correctly aligned is unknown. Three possibilities are shown at right. The ends may be positioned by base pairing with the opposite DNA strand, by base stacking with the adjacent residue, or by noncovalent interactions with the enzyme.

Because the enzyme does not form covalent bonds with the 3'-DNA termini, the basis for the alignment of the 3'-ends within the cleavage complex is not apparent. Three major possibilities exist (Fig. 1). First, the 3'-termini may be positioned for religation by base pairing to their complementary DNA strands. In support of this possibility, the 3'-terminal residues are located within the duplex portion of the cleaved helix following scission (18). Second, the 3'-termini may be positioned for religation by base stacking to their adjacent residues. In some cases, the energies of base stacking interactions approach those of base pairing (31). Third, the 3'-termini may be positioned by noncovalent interactions with topoisomerase II. This possibility is consistent with modeling studies (based on the crystal structure of yeast topoisomerase II) and mutagenesis experiments, which suggest that there are interactions between specific amino acids and the 3'-termini generated by cleavage (32, 33).

To distinguish between the above, the ability of human topoisomerase IIalpha to religate a series of oligonucleotide substrates with altered base pairing or base stacking at their 3'-DNA termini was determined. The strategy underlying these religation experiments is as follows. If the 3'-DNA termini are positioned primarily by base pairing or base stacking, any modification that disrupts these interactions should significantly decrease the rate of religation mediated by topoisomerase II. In contrast, if the 3'-DNA termini are positioned primarily by noncovalent interactions between the enzyme and the terminal base, only modifications that remove or alter this base should inhibit religation. Modifications that disrupt base pairing or base stacking without affecting the 3'-terminal base should have marginal, if any, effects.

Cleavage and Religation of Substrates Containing a Single DNA Lesion-- The initial sequence utilized for cleavage and religation studies was a double-stranded 40-mer derived from plasmid pBR322 (18, 27). This sequence contained a single strong cleavage site for topoisomerase II. The central portion of the oligonucleotide (with points of cleavage denoted by arrows) is shown in Fig. 2. The nucleotide at the -1 position relative to the point of cleavage becomes the 3'-terminal residue following scission. The nucleotide at the +5 position on the opposite strand is the residue that base pairs with the 3'-terminal base. To disrupt base pairing of the 3'-terminus without precluding protein interactions with the terminal base, an abasic site or a mismatched base was placed at the +5 position of the oligonucleotide. To disrupt both base pairing and potential protein interactions with the 3'-terminal base, an abasic site or a mismatched base was placed at the -1 position.


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Fig. 2.   Effects of DNA lesions that alter the 3'-termini on topoisomerase II-mediated DNA cleavage. Abasic sites (AP) or deoxyuridine (dU) residues were incorporated into the top (left panel) or bottom (right panel) strands of the 40-base pair pBR322 sequence at the indicated positions. Arrows represent the points of topoisomerase II-mediated cleavage, with open arrows and asterisks denoting the 32P-labeled DNA strand. Levels of enzyme-mediated DNA cleavage were calculated relative to that of the wild-type sequence (WT, set to 1 for each strand). Data represent the averages of >= 3 independent experiments, and S.E. are indicated by error bars.

As a prelude to religation studies, the ability of human topoisomerase IIalpha to cleave the modified oligonucleotide substrates was examined (Fig. 2). Consistent with previous studies (23, 29, 34), the incorporation of an abasic site or a base mismatch immediately outside the points of DNA cleavage decreased equilibrium levels of scission (Fig. 2). However, even in the most dramatic case (-1 AP), levels of scission were still high enough to reproducibly monitor rates of religation.

Fig. 3 depicts religation mediated by human topoisomerase IIalpha for the top and bottom strands (left and right panels, respectively) of the pBR322 oligonucleotide substrate. Similar results were obtained for both strands. In all cases, rates of religation for oligonucleotides modified at the +5 position (either abasic sites or uracil mismatches) were comparable to or faster than that of wild-type. This finding indicates that base pairing is not used to align the 3'-termini for religation.


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Fig. 3.   Effects of DNA lesions that alter the 3'-termini on topoisomerase II-mediated DNA religation. Abasic sites (AP) or deoxyuridine (dU) residues were incorporated into the top (left panel) or bottom (right panel) strands of the pBR322 sequence at the indicated positions. Arrows represent the points of topoisomerase II-mediated cleavage, with open arrows and asterisks denoting the 32P-labeled DNA strand. Religation is shown for the wild-type substrate (WT, ) and oligonucleotides containing an apurinic site (AP) at the -1 (triangle ) or +5 () position or a deoxyuridine residue (dU) at the -1 (black-triangle) or +5 (black-square) position on the top (left panel) or bottom (right panel) DNA strand. At time 0, levels of DNA cleavage for each oligonucleotide were set to 100%. Rates of religation were determined by monitoring the loss of cleaved substrate. Data represent the averages of >=  3 independent experiments, and S.E. are indicated by error bars.

In marked contrast to the results observed with the +5 modifications, the placement of an abasic site at the -1 position dramatically decreased the rate of religation. The presence of a uracil at the -1 position (which still allows base stacking (35, 36) but has the potential to alter protein-DNA interactions) also diminished the religation rate, albeit to a lesser extent than that observed with substrates that lacked a -1 base. Taken together, these results indicate that the presence of the base at the -1 position is critical to the alignment of the 3'-DNA termini. However, they cannot distinguish whether the termini are positioned by base stacking interactions or by noncovalent topoisomerase II-DNA interactions.

Cleavage and Religation of Substrates Containing Two DNA Lesions-- The experiments described above have one potential caveat. Because the initial amount of scission observed with the -1 abasic site oligonucleotides was low (~10% of that for the wild-type substrate), it is possible that religation of these modified oligonucleotides does not completely reflect the capabilities of the type II enzyme. Therefore, a second abasic site was incorporated into the pBR322 substrate to increase the starting level of DNA cleavage. This second lesion was located within the 4-base cleavage overhang at the +2 position (Fig. 4). Previous work indicates that the incorporation of abasic sites between the points of cleavage stimulates topoisomerase II-mediated DNA scission without affecting rates of religation (23). As seen in Fig. 4, following the inclusion of a +2 lesion in the top strand, levels of cleavage of the -1 abasic site oligonucleotide approached that of wild-type. Cleavage of a comparable +5,+2 bottom strand abasic substrate exceeded that of the unmodified substrate.


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Fig. 4.   Effects of dual abasic lesions on topoisomerase II-mediated DNA cleavage and religation of pBR322 oligonucleotides with altered 3'-termini. Abasic sites were incorporated into the topoisomerase II cleavage site of the pBR322 oligonucleotide at the positions indicated. Arrows represent the points of topoisomerase II-mediated cleavage, with the open arrow and asterisk denoting the 32P-labeled DNA strand. The left panel shows topoisomerase II-mediated DNA cleavage. Values for the dual abasic oligonucleotides (solid bars) were calculated relative to that of the wild-type substrate (WT, set to 1) and are compared with those of singly modified oligonucleotides (hatched bars). The right panel shows rates of religation of the wild-type substrate (WT, ) and oligonucleotides that contained a lesion that affected the 3'-terminus and an internal abasic lesion; -1,+2 (open circle ) and +5,+2 (). At time 0, levels of DNA cleavage for each oligonucleotide were set to 100%. Data represent the averages of >= 3 independent experiments, and S.E. are indicated by error bars.

Similar to results obtained with the single lesion substrates, the oligonucleotide that eliminated base pairing without removing the 3'-terminal base (i.e. the +5,+2 abasic substrate) displayed wild-type rates of religation (Fig. 4).1 Furthermore, the ability of topoisomerase II to religate the oligonucleotide that eliminated potential base stacking or protein interactions with the 3'-terminal base (i.e. the -1,+2 substrate) was severely inhibited (Fig. 4).2

To confirm the results found with the pBR322 substrate, cleavage and religation of a second oligonucleotide was characterized (Fig. 5). This second substrate was derived from a region of the MLL oncogene located proximal to a leukemic breakpoint at chromosomal band 11q23 (28, 29). Once again, oligonucleotides included two abasic sites to avoid potential problems associated with the poor cleavage of single lesion substrates.


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Fig. 5.   Effects of dual abasic lesions on topoisomerase II-mediated DNA cleavage and religation of MLL oligonucleotides with altered 3'-termini. Abasic sites were incorporated into the topoisomerase II cleavage site of a 40-base pair MLL oligonucleotide at the positions indicated. Arrows represent the points of topoisomerase II-mediated cleavage, with the open arrow and asterisk denoting the 32P-labeled DNA strand. The left panel shows topoisomerase II-mediated cleavage. Values for the dual abasic oligonucleotides were calculated relative to that of the wild-type substrate (WT, set to 1). The right panel shows rates of religation of the wild-type substrate (WT, ) and oligonucleotides that contained a lesion that affected the 3'-terminus and an internal abasic lesion; -1,+4 (open circle ) and +5,+2 (). At time 0, levels of DNA cleavage for each oligonucleotide were set to 100%. Data represent the averages of >= 3 independent experiments, and S.E. are indicated by error bars.

Cleavage levels of both the +5,+2 and -1,+4 dual abasic oligonucleotides exceeded that of wild-type (Fig. 5). As above, religation of the 3'-DNA terminus appeared to be unaffected by the loss of base pairing in the +5,+2 abasic substrate. However, religation was drastically impaired when the 3'-base was absent in the -1,+4 abasic substrate (Fig. 5). Similar results were observed for the top strand 3'-terminus when corresponding +5,+2 and -1,+4 oligonucleotides were employed (not shown).

Effects of the 3'-Terminal Base on the DNA Cleavage/Re-ligation Equilibrium of Topoisomerase II-- The above experiments provide strong evidence that the 3'-terminal base is important for topoisomerase II-mediated DNA religation. However, the poor rates of religation observed for substrates that lacked this base were determined under conditions in which topoisomerase II catalyzed a unidirectional rejoining event. Therefore, an "approach to equilibrium" experiment was carried out to determine whether the lack of a 3'-terminal base also impairs religation under conditions in which topoisomerase II catalyzes a complete DNA cleavage/religation cycle.

The time required for topoisomerase II to reach its cleavage/religation equilibrium reflects both the rates of cleavage and religation. Under normal conditions, the rate of religation appears to be limiting (37). Therefore, if the loss of the 3'-terminal base inhibits DNA religation, the time it takes for the enzyme to establish a DNA cleavage/religation equilibrium should be considerably longer than required for the comparable wild-type substrate. As seen in Fig. 6, human topoisomerase IIalpha establishes an equilibrium with the wild-type pBR322 oligonucleotide within 2 min. A similar time to establish equilibrium was observed for the +5,+2 abasic pBR322 oligonucleotide, which disrupts base pairing without removing the 3'-terminal base and displays wild-type rates of religation (Fig. 4). In contrast, when the -1,+2 abasic oligonucleotide (which eliminates the 3'-terminal base and displays dramatically lower rates of religation (Fig. 4)) was employed, the enzyme had yet to reach equilibrium by 20 min. This finding supports the conclusion that the elimination of the 3'-terminal base impairs the religation activity of topoisomerase II.


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Fig. 6.   Effects of lesions that alter the 3'-termini on the time required for topoisomerase II to reach DNA cleavage/religation equilibrium. Abasic sites were incorporated into the topoisomerase II cleavage site of the pBR322 oligonucleotide at the positions indicated. Arrows represent the points of topoisomerase II-mediated cleavage, with the open arrow and asterisk denoting the 32P-labeled DNA strand. Time courses for the appearance of topoisomerase II-mediated DNA breaks are shown for the wild-type (WT, ), -1,+2 abasic (open circle ), and +5,+2 abasic () substrates. Levels of cleavage were relative to that of wild-type, which was set to 1 at equilibrium. Data are representative of three independent experiments.

Effects of 3'-Terminal Base Stacking on Topoisomerase II-mediated Religation-- The above results establish the importance of the 3'-terminal base in topoisomerase II-mediated DNA religation and rule out the requirement for terminal base pairing in this process. However, they do not address the potential contributions of base stacking or protein-DNA interactions in aligning the 3'-terminal residue for religation. A prominent role for base stacking seems unlikely, because all of the cleavage substrates used in the present study contain terminal thymine-guanine sequences. Of the possible nearest neighbors in DNA, thymine-guanine steps exhibit one of the weaker base stacking energies (31, 35).

The above notwithstanding, an additional religation experiment was carried out to determine directly whether the 3'-DNA terminus is aligned for topoisomerase II-mediated re-ligation by base stacking interactions. This experiment utilized an oligonucleotide substrate that replaced the thymine at the -2 position with an abasic site. This substitution eliminates base stacking between the 3'-terminal residue and its nearest neighbor, but preserves terminal base pairing and potential interactions with the enzyme.

As seen in Fig. 7, human topoisomerase IIalpha cleaved the -2 abasic substrate with an efficiency that was similar to that of wild-type. Rates of DNA religation observed for the abasic oligonucleotide also were comparable with the unmodified sequence. This finding argues strongly against an important role for base stacking in aligning the 3'-DNA termini for religation.


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Fig. 7.   Effects of base stacking interactions on topoisomerase II-mediated DNA religation. An abasic sites was incorporated into the topoisomerase II cleavage site of a 40 base pair pBR322 oligonucleotide at the position indicated. Arrows represent the points of topoisomerase II-mediated cleavage, with the open arrow and asterisk denoting the 32P-labeled DNA strand. Religation is shown for the wild-type substrate (WT, ) and an oligonucleotide containing an apurinic site at the -2 (open circle ) position on the top DNA strand. At time 0, levels of DNA cleavage for each oligonucleotide were set to 100%. Rates of religation were determined by monitoring the loss of cleaved substrate. The inset shows topoisomerase II-mediated DNA cleavage. Values for the abasic oligonucleotide (-2) were calculated relative to that of the wild-type substrate (WT, set to 1). Data represent the averages of two independent experiments, and S.E. are indicated by error bars.

In conclusion, the DNA religation activity of topoisomerase II is critical to its physiological functions and its role as a target for anticancer agents (4-6, 8, 12-15). Yet, the mechanism of this process is not completely understood. A previous study demonstrated that the enzyme utilizes covalent protein-DNA interactions to align the 5'-DNA termini for religation (23). However, because topoisomerase II does not form covalent bonds with the 3'-termini, it was not clear how these unattached ends are positioned correctly for DNA religation. The present work indicates that efficient religation requires the presence of the 3'-terminal base. The proper alignment of this base does not depend on either interstrand base pairing or intrastrand base stacking interactions. Based on these findings, we propose that the 3'-termini are properly positioned for DNA religation by noncovalent contacts between topoisomerase II and the terminal base.

    ACKNOWLEDGEMENTS

We thank to Dr. P. S. Kingma for helpful discussions and to Dr. J. M. Fortune and K. D. Bromberg for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Grants GM33944 and GM53960 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Trainee under National Institutes of Health Grant 5 T32 CA09385.

|| To whom correspondence should be addressed: Dept. of Biochemistry, 654 Robinson Research Bldg., Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Tel.: 615-322-4338; Fax: 615-343-1166; E-mail: osheron@ctrvax.vanderbilt.edu.

Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M100197200

1 For all oligonucleotides that contained dual abasic lesions, rates of religation were determined for corresponding substrates that contained single +2 abasic sites (pBR322 sequences) or +4 abasic sites (MLL sequences). In all cases, rates were comparable with those of the wild-type oligonucleotides.

2 The DNA religation assays used for these experiments were based on trapping a kinetically competent topoisomerase II-DNA cleavage complex formed in the presence of CaCl2. Comparable results were obtained in parallel experiments that established DNA cleavage/re-ligation equilibria in the presence of MgCl2 and induced religation by lowering the reaction temperature to 0 °C.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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