HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 30, 26865-26871, July 26, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/26865    most recent M204641200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wilson-Sali, T. Articles by Hsieh, T.-s. Search for Related Content PubMed PubMed Citation Articles by Wilson-Sali, T. Articles by Hsieh, T.-s. Social Bookmarking                      What's this?

Generation of Double-stranded Breaks in Hypernegatively Supercoiled DNA by Drosophila Topoisomerase III, a Type IA Enzyme^*

Tina Wilson-Sali and Tao-shih Hsieh

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27704

Received for publication, May 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Drosophila topoisomerase (topo) III is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5'-end of DNA. We show here that a purified preparation of topo III is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 °C, maximal cleavage occurs between 23 and 30 °C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo III possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo III cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo III may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Processes such as replication, transcription, chromosome segregation, and recombination require the activities of a class of enzymes called topoisomerases (topos).¹ Topoisomerases are classified as either type I or type II enzymes, depending on overall structure and mechanism (reviewed in Refs. 1-3). These enzymes work by going through two cycles of transesterification. The first one is initiated by the active-site tyrosine to form a phosphodiester bond with the 5'- or 3'-DNA phosphate at the transient break site, and the second one is the reversal in which the 3'- or 5'-hydroxyl at the break reforms the DNA backbone bond, thus regenerating the free tyrosine in the enzyme. The type I enzymes are usually monomers that work by making a reversible single-stranded break in the DNA backbone, whereas type II enzymes are dimeric in structure and thus can introduce a transient double-stranded break. Passage of an intact strand(s) through the broken strand(s) followed by religation of the broken ends completes the topoisomerase reaction cycle and affects the topological transformation in DNA. The type I enzymes are further divided into the IA and IB families, with the Escherichia coli  protein as the prototypical type IA enzyme (4, 5). The first eukaryotic type IA topoisomerase was discovered in yeast through a genetic screen for suppression of recombination among repeated sequences (6), and more type IA enzymes (the topo III enzymes) subsequently have been identified in other higher eukaryotes (7-11). Prokaryotes like E. coli and lower eukaryotes such as yeast each possess one topo III enzyme, whereas the metazoans like worms, fruit flies, mice, and humans contain two topo III isoforms, designated topo III and topo III.

Although much is known about topo I and topo II, the precise cellular function of topo III remains to be elucidated. Biochemical and genetic data argue that, unlike topo I and topo II, the topo III enzymes may not contribute significantly to regulating the cellular levels of DNA supercoiling (12). For example, E. coli topo III is only one-quarter as active as E. coli topo I in the relaxation of negatively supercoiled DNA (13). The ability of Topo III to relax supercoils can be stimulated, however, when plasmid substrates contain a region of single-stranded DNA. This can be accomplished by incubating the reaction at high temperature to partially denature the plasmid, by using a substrate that contains a single-stranded loop, or by using a substrate that is hypernegatively supercoiled (8, 11, 13, 14).

Although the precise function of topo III is not yet known, its cellular importance is attested by the wide range of phenotypes presented by various topo III mutants. Both E. coli topB and Saccharomyces cerevisiae top3-null mutants have increased levels of recombination, particularly within repeated DNA sequences (6, 15-17). The hyper-recombination phenotype of topo III mutants suggests that it has a critical role in regulating recombination. An essential function of type IA enzymes in resolving recombination intermediates may account for the chromosome segregation defects observed in bacterial top1 and top3 mutants (18) and in yeast top3 mutants (19-21). In addition, yeast topo III has an important function in the intra-S checkpoint and in the repair of lesions caused by a wide range of DNA-damaging agents (22). Transgenic mouse studies likewise have demonstrated important functions for the topo III isozymes in mammals. Intriguingly, although the topo III knockout mouse is viable, it has a shortened lifespan relative to its heterozygous mutant and homozygous wild-type littermates, and the topo III knockout mouse dies in utero (23, 24). Such experiments demonstrate that in all systems studied to date, loss of topo III function cannot be completely compensated by any other topoisomerase in the cell, including another topo III isoform.

The topo III enzymes interact genetically, physically, and functionally with members of the RecQ family of DNA helicases (25-28). Humans possess five RecQ homologs: RecQL, BLM, WRN, RTS, and RecQ5. Mutations in BLM, WRN, and RTS lead to Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson syndrome, respectively (reviewed in Ref. 29). The human topo III and BLM proteins have been shown to interact physically, with BLM helping topo III localize to nuclear PML bodies where they act to prevent sister-chromatid exchange (30). In addition, RecQ5 also has been shown to interact with topo III, which interacts with chromosomes specifically during M-phase (27, 31). Therefore, the topo III/RecQ partners have a demonstrated function in stabilizing genomes during mitotic and meiotic growth.

Although the interplay between the topo III and RecQ enzyme families provides interesting insight into the biological functions of these enzymes, much remains to be discovered about the basic biochemical properties of the topo III enzyme that are relevant for its in vivo roles. To that end, we describe here a reversible cleavage activity for Drosophila topo III and discuss the possible role such an activity might reveal about the biological function of the topo III enzyme.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Purification and Enzyme Assays-- Drosophila topo III was purified according to our published procedure with the following modification (11). Briefly, the previous procedure involved the fractionation of nuclear extract by column chromatography with Ni (II)-nitrilotriacetic acid-agarose, single-stranded DNA-agarose, and hydroxyapatite. We eliminated the single-stranded DNA-agarose step from our purification because it invariably incurs a significant loss of topo III. This is because of the high affinity of topo III for the single-stranded DNA (data not shown). The resulting protein has a purity comparable with the previous preparation, but an ~10-fold higher concentration (1 mg/ml). We have prepared an active site mutant of topo III and purified it using an identical procedure as that for the wild-type enzyme. The site-specific mutagenesis to convert the active site tyrosine at residue 332 to phenylalanine was described previously (32).

Standard topo III reactions were incubated for 1 h in 40 mM Hepes-KOH (pH 7.5) and 1 mM MgCl₂ at a temperature and concentration specified in the text. Purification of Drosophila topo I-ND423 also was reported previously (33). Standard topo I reactions were for 1 h at 30 °C in 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 10 mM MgCl₂, 100 mM NaCl, and 0.1 mM EDTA. All reactions were stopped by the addition of 10 mM EDTA, 0.2% SDS, and 0.15 mg/ml proteinase K. Samples were heated at 45 °C for 15 min before analysis by 1.2% agarose gel electrophoresis was performed. In the absence of proteinase K treatment, the protein-DNA complexes were resolved by running the samples in buffer containing 0.1% SDS.

Southern Blots-- Samples for Southern blot analysis were purified after topo III reaction by phenol extraction and ethanol precipitation. DNA was resuspended and dialyzed against TE buffer (10 mM Tris-HCl (pH 7.9) and 0.1 mM EDTA) and then digested with restriction enzyme (New England Biolabs). The digested material was purified by phenol extraction and ethanol precipitation and then resuspended in TE buffer. Samples were electrophoresed through a denaturing or non-denaturing agarose gel at a constant voltage of 20 V for 20-30 h. After denaturation and neutralization, samples were transferred to nitrocellulose and hybridized with radiolabeled probes: MAP1, 5'-ATCGTGGCCGGCATCAC-3' or MAP 3, 5-'GTCGCCATGATCGCGTA-3'. Prior to hybridization, oligonucleotide probes were 5'-end-labeled with T4 polynucleotide kinase (USB) and [-³²P]ATP (PerkinElmer Life Sciences). Unincorporated label was removed by passage of the samples over a BioRad Bio-Spin 6 column. Plasmid A/T content was plotted using MacVector 7.0 software (Kodak).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cleavage of Hypernegatively Supercoiled DNA with a Concentrated Preparation of Drosophila Topoisomerase III-- Our earlier experiments showed that at an enzyme/DNA molar ratio of 5:1, topo III can relax hypernegatively supercoiled DNA to a supercoiling level slightly higher than that of the native plasmid DNA (11). To test whether higher enzyme concentrations may affect further relaxation, we titrated topo III enzyme in the relaxation reactions at enzyme/DNA ratios ranging from 0.1:1 to 100:1 (Fig. 1A). The slight shift in mobility from the hypernegatively supercoiled DNA to the negatively supercoiled plasmid indicates similar levels of relaxation by topo III, as demonstrated previously. Interestingly, at ratios of 25:1 or greater (Fig. 1A, lanes 10-13), the majority of the hypernegatively supercoiled DNA was converted into primarily nicked but also linear species. At the greatest concentration of topo III tested (100:1, lane 13) nearly all of the substrate had been cleaved, but both the nicked and linear species can be detected in the reactions starting as low as 5:1 (lane 5). The nicked DNA can be distinguished from fully relaxed DNA both by the absence of a ladder of topoisomers (which generally is observed with fully relaxed DNA under these electrophoretic conditions) and by the lack of a change in mobility when electrophoresed in the presence of ethidium bromide (data not shown). No nicking of the DNA was observed at molar ratios of 25:1 or greater when the hypernegatively supercoiled DNA was incubated with a purified active site Y332F mutant of topo III (data not shown). In contrast, incubation of hypernegatively supercoiled DNA with another type I topoisomerase, Drosophila topo I, at a ratio of 50:1 resulted in full relaxation of the hypernegatively supercoiled DNA (Fig. 1B).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Titration of Drosophila topoisomerase III in reactions with hypernegatively supercoiled DNA. A, hypernegatively supercoiled DNA was incubated with increasing amounts of purified Drosophila topo III under standard conditions for 1 h at 37 °C. Lanes 1-13 indicate enzyme/DNA molar ratios ranging from 0.1:1 to 100:1. Negatively supercoiled (NSC) and hypernegatively supercoiled (HNSC) markers were run as indicated. B, hypernegatively supercoiled plasmid DNA was incubated with Drosophila topo I ND-423 (enzyme/DNA molar ratio of 50:1) under standard conditions for 1 h at 30 °C. Samples were electrophoresed in the presence of 0.75 µg/ml ethidium bromide. Fully relaxed and hypernegatively supercoiled DNA markers were run as shown.

One interesting feature of the topo III reaction is that the nicked and linear cleavage products can be generated by stopping the reaction with proteinase K and either 10 mM EDTA, 0.2% SDS, or both (data not shown). When proteinase K is excluded from the stop mix the DNA fails to resolve through the gel, suggesting covalent complex formation between the protein and the DNA (Fig. 2A). The DNA in these untreated samples can be resolved as distinct bands, however, if the samples are electrophoresed through an agarose gel containing SDS (Fig. 2B, left panel). These are likely the covalent protein-DNA adducts, as demonstrated before for the topo II cleavage reactions (34). The protein-DNA complexes can be transferred to a nitrocellulose membrane, and binding of topo III to the nicked and linear DNA fragments can be confirmed by Western blot (Fig. 2B, right panel).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Drosophila topoisomerase III is covalently linked to the nicked and linear cleavage products. A, Drosophila topo III was incubated with hypernegatively supercoiled DNA under standard conditions for 1 h at 37 °C. Half of the reaction was treated with proteinase K in the stop mix (+ lane) as described under "Experimental Procedures" while the other half of the reaction was stopped without undergoing proteinase K treatment ( lane). Hypernegatively supercoiled (HNSC), nicked, and linear DNA markers were run as shown. B, two sets of samples like those described in the legend to Fig. 2A were resolved on a gel containing 0.1% SDS. The gel was cut in half, and one set of samples was stained with ethidium bromide (left panel) while the other set was transferred to a nitrocellulose filter and analyzed by Western blot with antibody against Drosophila topo III (right panel). Only the nicked and linear DNA in the untreated sample contain a linked topo III moiety. HNSC, nicked, and linear DNA markers were run as shown.

More Extensive Cleavage of the Hypernegatively Supercoiled DNA Occurs at Lowered Temperature-- It is interesting to note that in the previous experiment with topo III the majority of the cleaved material was in the form of nicked DNA, but there does appear to be a significant amount of linear DNA as well. To further investigate this cleavage phenomenon, Drosophila topo III was incubated with hypernegatively supercoiled DNA at various temperatures ranging from 0 to 50 °C at an enzyme/DNA ratio of 100:1 (Fig. 3). Although the appearance of nicked and linear DNA was observed at all temperatures below 50 °C, more extensive cleavage of the substrate occurred at 30, 23, and 15 °C, with the maximal cleavage occurring between 23 and 30 °C. This is in contrast to the relaxation activity of topo III, which is optimal at 37-45 °C. The distinct banding pattern of the subunit-length DNA fragments in the cleavage reactions is similar to that of a topo II-mediated reaction. This result suggests that topo III may cleave both strands in a single-stranded bubble in the hypernegatively supercoiled DNA.

View larger version (60K): [in this window] [in a new window]   Fig. 3.   Extensive cleavage of hypernegatively supercoiled DNA occurs at lowered temperatures. Drosophila topo III was incubated with hypernegatively supercoiled DNA (100:1 molar ratio) under standard buffer conditions for 1 h at 50, 37, 30, 23, 15, 4, and 0 °C (lanes 1-7, respectively). Nicked, linear, negatively supercoiled (NSC), and hypernegatively supercoiled (HNSC) plasmid markers were run as indicated.

The Cleavage Products Are Stable Over Time and Reversible-- The protein linkage of the nicked and linear cleavage products suggests that the subunit-length DNA fragments are not generated by an endonuclease. To further rule out the possibility that the DNA cleavage products were generated by a nuclease contaminating the enzyme preparation, we investigated the stability of the products over time as well as whether or not the cleavage events were reversible (Fig. 4).

View larger version (83K): [in this window] [in a new window]   Fig. 4.   The ladder of DNA cleavage products is stable over time and reversible. A, Drosophila topo III was incubated with hypernegatively supercoiled DNA at 23 °C under standard buffer conditions. Aliquots of the reaction were removed and stopped after 1 min, 30 min, 1 h, 2 h, 5 h, and 20 h of incubation (lanes 1-6, respectively). B, Drosophila topo III was incubated with hypernegatively supercoiled DNA at 23 °C under standard buffer conditions. After 1 h, separate aliquots were removed and either stopped (lane 1) or subjected to an increase in the NaCl concentration (to 1 M, lane 2; to 0.5 M, lane 3), or the MgCl2 concentration (to 10 mM, lane 4) for 1 h at 23 °C, or they were incubated at 70 °C for 10 min (lane 5) before the aliquots were stopped. As a control, an aliquot was also continued under standard conditions for an additional 1 h at 23 °C (lane 6). HNSC, hypernegatively supercoiled; NSC, negatively supercoiled.

Topo III enzyme was incubated with hypernegatively supercoiled DNA at 23 °C (enzyme/DNA ratio of 100:1). Aliquots were removed and stopped at time points ranging from 1 min to 20 h (Fig. 4A, lanes 1-6). The time course reveals that the ladder of cleavage products can be observed within the first minute of incubation and persists 20 h later. Such stability argues against nuclease contamination. Additionally, aliquots were removed after 1 h of incubation and either heated at 70 °C for 10 min or subjected to an increase in either NaCl (to 0.5 or 1 M) or MgCl₂ (to 10 mM) concentration and incubated for an additional hour at 23 °C before the reactions were stopped (Fig. 4B, lanes 1-6). Both the heat and salt treatments resulted in a depletion of cleavage products and a concomitant appearance of supercoiled DNA, which migrated at approximately the same mobility as the hypernegatively supercoiled starting material. Such an efficient reversibility illustrates a hallmark of topoisomerase-mediated DNA cleavage, namely that religation of broken DNA ends can be induced by shifts in either ionic or temperature conditions. The reversibility and the stability over prolonged incubation demonstrate that the cleavage ladder most likely is not generated by a contaminating nuclease but rather that topo III itself possesses a reversible endonucleolytic cleavage activity.

The Cleavage Activity of Drosophila Topoisomerase III Is Specific for Substrates That Contain a Single-stranded Region-- We found previously that Drosophila topo III can relax hypernegatively supercoiled plasmid but not natively isolated, negatively supercoiled DNA (11). To investigate the substrate specificity for the cleavage activity, topo III was incubated with seven different hypernegatively supercoiled substrates as well as their corresponding negatively supercoiled parental plasmids (Fig. 5, A and B). All seven hypernegatively supercoiled substrates, ranging in size from 3 to 16 kb, could be converted into a ladder of cleavage products, whereas none of the negatively supercoiled plasmids appear to be acted upon by topo III. This is consistent with previous reports regarding the affinity of topo III for highly unwound substrates.

View larger version (70K): [in this window] [in a new window]   Fig. 5.   Topoisomerase III exhibits substrate specificity with regard to its cleavage activity. Drosophila topo III was incubated under standard conditions for 1 h at 23 °C with seven separate hypernegatively supercoiled substrates containing different sequences (A) and their corresponding negatively supercoiled parental plasmids (B). Absence and presence of enzyme are indicated with  and + signs, respectively. C, M13 single-stranded circle (ss circle) was incubated with Drosophila topo III at 37 °C, and aliquots were removed and stopped after 15 min, 30 min, 1 h, 2 h, and 4 h (lanes 1-5, respectively). After incubation for 1 h, an aliquot was removed and incubated at 70 °C for 10 min before the reaction was stopped (lane 6). HNSC, hypernegatively supercoiled; NSC, negatively supercoiled.

Although the exact structure of hypernegatively supercoiled plasmid is unknown, it is predicted to be unwound to such a degree that single-stranded regions are exposed. To test the ability of topo III to cleave a single-stranded substrate, we incubated the enzyme with M13 single-stranded circular DNA (Fig. 5C). A time course demonstrates that the circle could be converted into linear DNA as well as a smear of smaller cleavage products and that these products persisted after 4 h of incubation. At early time points, a similar cleavage pattern was produced when the single-stranded circle was treated with DNase I, but the DNA was further degraded over time (data not shown). When an aliquot of the topo III reaction was removed after 1 h and heated at 70 °C for 10 min before the reaction was stopped, the smear condensed to fragments of higher molecular weight (Fig. 5C, lane 6), suggesting partial reversion and religation of the cleavable complexes. Taken together, these experiments demonstrate that topo III is specific for highly unwound or single strand-containing substrates.

Southern Blot Analysis Reveals the DNA Cleavage Is Concentrated in Highly A/T-rich Regions-- In order to determine the sites of cleavage for topo III, we probed the cleavage fragments of two hypernegatively supercoiled substrates, pBR322 and pKK405, using an indirect end-labeling method (Fig. 6). pKK405 was constructed by insertion of a 1.35-kb fragment containing the T4 phage ori(uvsY) origin of replication into pBR322 (35, 36). The hypernegatively supercoiled substrates were reacted with topo III at 37 °C and at an enzyme/DNA ratio of 25:1 to minimize the number of cleavage events per DNA molecule. The DNA samples were then purified and linearized with BamHI before electrophoresis through a denaturing agarose gel and subjection to Southern blot analysis (Fig. 6A).

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Mapping the sites of DNA cleavage by Southern blot. Hypernegatively supercoiled pBR322 (lane 1) and pKK405 (lane 2) samples were cut with topo III, purified, and linearized with BamHI. After electrophoresis through a denaturing (A) or non-denaturing (B) agarose gel, samples were subjected to Southern blot analysis using 32P-labeled probes (MAP 1 and MAP 3) that allow for detection of cleavage along the full length of both strands. C, representation of the linearized plasmids. The MAP 1 and MAP 3 probes are shown adjacent to the strands to which they anneal. The locations of topo III cleavage within the DUE and T4 ori regions, as determined from the Southern blots, are indicated by brackets, and the range of cleavage is numbered.

The strategy for mapping the cleavage sites is shown in Fig. 6C. The MAP1 and MAP3 probes annealed within 15-20 base pairs of the BamHI ends, allowing examination of topo III cleavage along the full length of both strands. The presence of full-length linear DNA on the Southern blot shows that a substantial amount of the plasmid substrate was not cleaved by topo III and therefore that the reaction conditions were such as to minimize the number of cleavage events per DNA molecule. Although the presence of smears in the denaturing gel suggests that both pBR322 and pKK405 plasmids were cut by topo III along their full length, both the MAP1 and MAP3 Southern blots demonstrate that the cleavage was concentrated primarily within distinct regions. In these indirect end-labeling experiments, the location of the cleavage sites can be determined simply by measuring the size of the cleavage products. The Southern blots reveal that pBR322 was cut mainly within a DNA-unwinding element, or DUE region, located on the plasmid between the ampicillin-resistance gene and the Col E1 origin of replication, whereas pKK405 was cut both within the DUE region and within the T4 ori(uvsY) insert. The range of cleavage was further refined by digesting the topo III-cut DNA with restriction enzymes closer to these DUE and T4 ori regions and then probing with radiolabeled oligonucleotides specific for these ends (data not shown). The locations of the cleavage sites on the plasmids as determined by these Southern blots are indicated by brackets and numbered in Fig. 6C. A comparison of the cleavage regions determined by the Southern blot analysis to a plot of the base composition of these plasmids reveals that the DUE and T4 ori cleavage sites fall within highly A/T-rich sequences that peak at around an 80% A/T content (Fig. 7).

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Plot of the A/T composition of pBR322 and pKK405. The percent A+T within pBR322 and pKK405 was plotted as a function of position using MacVector 7.0 software. The locations of topo III cleavage, as determined by Southern blot analysis, are indicated by arrows, and the range of cleavage is numbered.

Interestingly, the MAP1 and MAP3 probes reveal cleavage on both strands within the DUE region for pBR322. Additionally, MAP1 reveals cleavage within the DUE for pKK405, whereas MAP3 reveals cleavage within the T4 ori region. There are two possible explanations for the pKK405 results; either there is a preference with regard to the strand that was cleaved, or double-stranded breaks were made in both areas, and only the one closest to the probe was revealed. In order to distinguish between these two possibilities, the samples were run through a non-denaturing agarose gel and blotted with MAP1 and MAP3 as before. The non-denaturing Southern blot confirms that both pBR322 and pKK405 experienced double-stranded breaks within the DUE region, while pKK405 also experienced double-stranded breaks within the T4 ori(uvsY) insert (Fig. 6B). This suggests that in the single-stranded bubble formed within highly A/T-rich regions of hypernegatively supercoiled DNA, topo III is competent to cleave either one (leading to nicked species) or both strands (leading to linear species) in these bubble regions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we describe a reversible cleavage activity for the DNA topoisomerase III enzyme, which has an exquisite specificity for hypernegatively supercoiled DNA. Although all type I topoisomerases make transient single-stranded breaks within DNA, we demonstrate that Drosophila topoisomerase III, a type I enzyme, can generate extensive but reversible double-stranded breaks within a hypernegatively supercoiled DNA substrate by nicking two complementary strands within close proximity. This double-stranded cleavage activity is observed only with substrates that are more unwound than natively isolated plasmid, most likely because such hypernegatively supercoiled substrates contain single-stranded bubble regions. Southern blot analysis maps the sites of cleavage primarily to regions in the plasmid that are highly A/T-rich. Presumably, these would be the first regions to denature in a plasmid under supercoiling stress.

At high enzyme/DNA ratios, the predominant topo III reaction products at 37 °C are nicked and linear DNA. This activity appears to be unique to topo III; a similar experiment performed with Drosophila topo I results in full relaxation rather than cleavage. The DNA cleavage reaction by topo III has a dramatic temperature dependence. Lowering the temperature of the reaction by only 5-10 °C results in formation of an extensive ladder of lower molecular weight cleavage products. The sharp resolution of the bands within the ladder suggests not only that each band is a product of at least four separate single-stranded cleavage events by topo III but also that there is a sequence preference for each cleavage event. The mechanistic basis for the difference in the optimal temperature for the cleavage versus relaxation activities is as yet unknown. It is interesting to note, however, that the cleavage activity of topo III occurs optimally at a temperature range (23-30 °C) that is more physiologically relevant for fruit flies than the relaxation activity (37-45 °C). This is in line with the fact that the relaxation activities of both Drosophila topo I and topo II are optimal at 30 °C (37, 38).

Interestingly, the cleavage products are generated when the reaction is stopped with proteinase K and either EDTA or SDS alone (as well as with both EDTA and SDS). Incubation with proteinase K alone does not result in DNA cleavage (data not shown). This is in contrast to either topo I or topo II, where cleavage must be induced with a strong protein denaturant such as SDS or alkali and requires the presence of a topoisomerase-targeting drug in order to generate extensive cleavage products. This difference suggests that although topo III, like the other topoisomerases, is able to bind DNA and generate reversible breaks, it may differ in terms of the stability of the cleavable complexes it forms with the DNA.

The cleavage of hypernegatively supercoiled DNA by topo III is very efficient. We can readily observe the generation of both nicked and linear DNA beginning at enzyme/DNA molar ratios of 5:1 along with the products of the relaxation reaction. In an interesting contrast, we have shown recently that plasmid-based R-loop and D-loop substrates are refractory to the relaxation activity of topo III but can be cleaved readily (32). Both the hypernegatively supercoiled and R-/D-loop DNAs contain an unwound segment and can be cleaved efficiently by topo III. These experiments support the idea that topo III possesses a reversible, structure-specific endonucleolytic activity that shows a preference for substrates containing single-stranded regions.

Such a structure-specific activity may have relevance concerning the biological functions of topo III. Topo III-mediated cleavage will generate a nascent 3'-hydroxyl end and, when coupled with a 3'- to 5'-DNA helicase activity like that from a RecQ homolog, can be used to initiate the formation of recombination intermediates. Interestingly, because the recombination intermediates likely have a structure with single-stranded character, cleavage by topo III followed by unwinding through the action of a DNA helicase can reverse or resolve the recombination intermediates. The reversal of the cleavage reaction can ligate the break and regenerate an intact DNA strand. Thereby, topo III and RecQ can function as a team to regulate the process of genetic recombination. The connection of a structure-specific endonuclease activity to recombination and repair is further implicated by the recent observation that Mms4/Mus81 has an overlapping and essential function with Sgs1/Top3 and forms a heterodimeric endonuclease (39, 40). MMS4 and MUS81 were among the genes originally obtained through a screen for synthetic lethals of sgs1 mutation in yeast, and they function together in genetic pathways involving recombination and repair (39). Interestingly, the Mms4/Mus81 heterodimer has a unique endonucleolytic activity that is specific for branched or forked substrates, suggesting a role for rescuing stalled replication forks (40). Direct biochemical evidence linking Mus81/Eme1, a homolog of Mus81/Mms4 in the fission yeast, to the resolution of Holliday junctions also has been shown recently (41). The biochemical basis for a role of topo III/RecQ in processing recombination intermediates and their relationships with other structure-specific enzymes like Mus81/Mms4 and Mus81/Eme1 will be an important area for future investigation.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM29006.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.

To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, Research Dr., Durham, NC 27710. Tel.: 919-684-6501; Fax: 919-684-8885; E-mail: hsieh@ biochem.duke.edu.

Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M204641200

	ABBREVIATIONS

The abbreviations used are: topo, topoisomerase; DUE, DNA- unwinding element.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				F. G. Harmon, J. P. Brockman, and S. C. Kowalczykowski RecQ Helicase Stimulates Both DNA Catenation and Changes in DNA Topology by Topoisomerase III J. Biol. Chem., October 24, 2003; 278(43): 42668 - 42678. [Abstract] [Full Text] [PDF]

				L. Wu and I. D. Hickson The Bloom's syndrome helicase stimulates the activity of human topoisomerase III{alpha} Nucleic Acids Res., November 15, 2002; 30(22): 4823 - 4829. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/26865    most recent M204641200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wilson-Sali, T. Articles by Hsieh, T.-s. Search for Related Content PubMed PubMed Citation Articles by Wilson-Sali, T. Articles by Hsieh, T.-s. Social Bookmarking                      What's this?