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. 2002 Dec 1;30(23):5087–5093. doi: 10.1093/nar/gkf659

Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro

Holger Stephan 1, Frank Grosse 1,a, Kent Søe 1
PMCID: PMC137972  PMID: 12466531

Abstract

Several studies have shown that human topoisomerase I (htopoI) cleaves in the vicinity of various DNA lesions and thereby forms covalent intermediates known as ‘cleavage complexes’. Such complexes are detrimental to cells if they are not repaired. Therefore, it is generally accepted that repair pathways must exist for such lesions. We have demonstrated that a htopoI cleavage complex can be recognized by a second topoisomerase I molecule and thereby perform a so-called htopoI ‘double cleavage’ in vitro. In addition, we found that the double cleavage reaction was stimulated by p53. Here we show that the double cleavage reaction results in the removal of the original htopoI cleavage complex and the generation of a single-stranded gap of ∼13 nt. This gap supports a sequence-dependent DNA recombination reaction mediated by the second htopoI molecule. Furthermore, we show that p53 strongly stimulates the recombination reaction. We suggest that this reaction may represent a novel p53-dependent topoisomerase I-induced recombination repair (TIRR) pathway for htopoI cleavage complexes.

INTRODUCTION

Human topoisomerase I (htopoI) is an essential enzyme that plays an important role in releasing torsional stress, which is built into DNA by replication and transcription (1–4). During the catalytic cycle htopoI becomes transiently and covalently attached to the 3′ end of one DNA strand by a phosphotyrosyl linkage. As a result of the cleavage a free 5′ OH group is generated. After the tension has been released the 5′ OH performs a nucleophillic attack on the phosphotyrosyl bond, the DNA is ligated, and the topoisomerase is released (5).

Besides removing torsional strain, htopoI cleaves in the vicinity of UV lesions in vitro (6). Thereby, htopoI is stabilized in the cleaved state, in a so-called ‘cleavage complex’. Later, cleavage was also found next to a variety of DNA lesions generated in vitro (7–9) as well as in vivo (10–12). For the majority of these lesions the cleavage complex is stabilized due to an inaccessibility of the 5′ OH group. Meanwhile, it is accepted that such lesions must be removed in order to avoid genomic instability or cell death (reviewed in 13). Consequently, a tyrosyl-DNA-phosphodiesterase (Tdp1) has been identified in yeast as well as in humans. This enzyme cleaves the bond between the topoisomerase and the DNA (9), but unfortunately only after the cleavage complex was processed by a protease (14). Yeast deficient in Tdp1 showed an increased sensitivity to the topoisomerase I inhibitor camptothecin (CPT), but it was also found that there must exist other pathways for the repair of topoisomerase I cleavage complexes (9). We were interested in investigating whether alternative repair pathways exist that can repair htopoI cleavage complexes.

Recently, we have demonstrated that a htopoI cleavage complex can be recognized by a second htopoI molecule, which cuts immediately upstream from the original htopoI cleavage complex in vitro (15). We called this phenomenon a htopoI ‘double cleavage’. Furthermore, we have found that this double cleavage reaction was strongly stimulated by the tumor suppressor protein p53 (16). We suggested that the htopoI double cleavage reaction may be the initial step in a removal of the original htopoI cleavage complex. In the present work we used a ‘suicide substrate’ to covalently trap htopoI onto DNA. The formation of such suicide complexes mimic the situation in vivo where a htopoI cleavage complex is stabilized in the vicinity of a DNA lesion (10–12). Here, we present data which show that the htopoI suicide cleavage complex is released from the DNA substrate upon cleavage by a second htopoI molecule. The generated gap serves as an entry site for a htopoI-mediated recombination reaction. Furthermore, we show that this recombination reaction is strongly stimulated by p53. Based on these data we suggest that the htopoI double cleavage reaction may represent a novel p53-dependent pathway for the repair of htopoI cleavage complexes by DNA recombination.

MATERIALS AND METHODS

Preparation of recombinant protein

Recombinant wild-type htopoI and mutant htopoIY723F was expressed in the baculovirus system and purified as de scribed previously (15). The recombinant mutant htopoIY723F baculovirus was a generous gift from Dr Philippe Clertant, University of Nice-Sophia Antipolis, Nice, France.

Murine His-tagged p53 (mHis-p53) was purified as follows. HiV insect cells (ITC Biotechnology, Heidelberg, Germany) were infected with the mHis-p53-expressing baculovirus clone for 46 h at 27°C. Approximately 6 × 108 cells were harvested by centrifugation. The pellet was resuspended in 4 pellet vol of lysis buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 20 mM imidazole, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 1 mM aprotinin and 1 mM leupeptin). The cells were lyzed using a Dounce Teflon homogenizor, spun at 48 000 g, and the resulting crude extract was loaded on a Ni-NTA agarose column (Qiagen) pre-equilibrated in wash buffer I (20 mM Tris–HCl, pH 8, 100 mM KCl, 20 mM imidazole, 2.5 mM β-mercaptoethanol). The column was washed with the same buffer and the protein was eluted with elution buffer I (20 mM Tris–HCl, pH 8, 100 mM KCl, 250 mM imidazole, 2.5 mM β-mercaptoethanol, 1 mM PMSF). The protein-rich fractions were loaded on a phosphocellulose column (P11, Whatman) pre-equilibrated with wash buffer II (100 mM potassium phosphate, pH 7.8, 1 mM EDTA and 2 mM β-mercaptoethanol) using FPLC (Amersham Pharmacia). The column was washed, and the protein was eluted with elution buffer II (300 mM potassium phosphate, pH 7.8, 1 mM EDTA and 2 mM β-mercaptoethanol). The fractions were analyzed by SDS–PAGE. p53-containing fractions were dialyzed overnight against storage buffer (20 mM HEPES–KOH, pH 7.8, 50 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT and 50% glycerol) and stored at –20°C until use.

Competition of the double cleavage reaction with mutant htopoIY723F

The radioactively labeled substrate L193s was produced by ligation of three oligonucleotides in such a way that the 32P radioactive label was placed at position 15 from the 3′ end and purified as described previously (15). The stoichiometry between L193s and htopoI was estimated to be 1:10 (L193s:htopoI).

Radioactively labeled L193s (1500 Bq) was or was not incubated with 500 ng of htopoI with the indicated amounts of mutant htopoIY723F in buffer A (15.5 mM HEPES–KOH, pH 7.9, 50 mM KCl, 34 mM NaCl, 6 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 18.5% glycerol) in 20 µl for 40 min at 37°C. The reaction was stopped by adding SDS to a final concentration of 0.7% SDS. An aliquot of 2 µl was taken from each sample and analyzed on a 7.5% SDS–PAGE followed by quantification using a phosphoimager and the ImageQuant software (Storm 860, Molecular Dynamics; Amersham Pharmacia Biotech). The remainder of each sample was incubated with 1 mg/ml proteinase K for 30 min at 37°C. The DNA was precipitated with 3 vol of 0.6 M LiCl in absolute ethanol for 30 min or overnight at 4°C. The pellet was redissolved in loading buffer [40% formamide, 25 mM Tris-borate pH 8.3, 0.5 mM EDTA, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue], heated to 95°C for 5 min, and analyzed on a 14% denaturing polyacrylamide gel (SequaGel; National Diagnostics). The gel was dried and analyzed using a phosphoimager and the software ImageQuant (as above).

Recombination experiments

The non-labeled DNA substrate L193s was purified as described previously (15). However, since the substrate was not radioactively labeled, the full-length product was identified by shadow casting on a thin-layer chromatography plate when irradiated with 254 nm UV light.

All OL26 recombination oligonucleotides were produced as follows: 10 pmol oligonucleotide (25 nt) (Purimex, Goettingen, Germany) was incubated with 20 U of terminal transferase for 30 min at 37°C as described by the supplier (New England BioLabs) in the presence of 1.85 × 106 Bq [α-32P]ddATP (111 TBq/mmol; Amersham Pharmacia Biotech). The reaction mixture was passed through a NAP-5 spin-column (Amersham Pharmacia Biotech). The run-through fraction was mixed with loading buffer (as above) and loaded on a 14% denaturing polyacrylamide gel. The correct band was excised, eluted, precipitated with 4 vol of 0.6 M LiCl in absolute ethanol on ice for 1 h, and redissolved in 30 µl of TE pH 7.5. The sequences of the oligonucleotides were: OL26, CAAAAAAAGACTTAGAAAAAAAAAAA; OL26-1T, TAAAAAAAGACTTAGAAAAAAAAAAA; OL26-3T, TTTAAAAAGACTTAGAAAAAAAAAAA; OL26-6T, TTTTTTAAGACTTAGAAAAAAAAAAA.

Recombination reactions

htopoI was incubated with non-labeled L193s and radioactively labeled OL26 as indicated in a total reaction volume of 50 µl in buffer B (13 mM HEPES–KOH, pH 7.9, 50 mM KCl, 8.5 mM NaCl, 6 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 11% glycerol) (see Figs 5 and 6) or in buffer C (14 mM HEPES–KOH, pH 7.9, 50 mM KCl, 17 mM NaCl, 6 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 13.5% glycerol) (see Fig. 4) or in buffer D (16.5 mM HEPES–KOH, pH 7.8, 50 mM KCl, 9 mM NaCl, 6 mM MgCl2, 1 mM DTT, 19% glycerol) (see Fig. 7). The reactions were stopped by incubation with 1.2 mg/ml trypsin for 30 min at 37°C. The DNA was precipitated with 4 vol of 0.6 M LiCl in absolute ethanol on ice for 1 h followed by centrifugation. The pellet was redissolved in 1-fold BamHI reaction buffer and 60 U BamHI (New England BioLabs) and incubated for 1 h at 37°C. The DNA was precipitated as above, redissolved in loading buffer, incubated for 5 min at 95°C, and loaded on a 14% denaturing polyacrylamide gel (as above). Subsequently, the gel was dried and exposed to a phosphoscreen and analyzed using a phosphoimager and the ImageQuant software (as above).

Figure 5.

Figure 5

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The recombination product is not produced by contaminating endonucleases. Autoradiogram of a 14% denaturing polyacrylamide gel. Fourteen femtomoles of 32P 3′-labeled OL26 was incubated for 60 min at 30°C with: lane 1, 11 pmol of htopoI + BamHI; lane 2, 500 pmol of L193s + BamHI; lane 3, 500 pmol of L193s + 11 pmol of htopoI; lane 4, 500 pmol of L193s + 11 pmol of htopoI + BamHI. The mobilities of the DNA fragments (nt) are indicated on the right-hand side.

Figure 6.

Figure 6

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The recombination reaction depends on sequence homology to the gap. (A) Autoradiogram of a 14% denaturing polyacrylamide gel. Fourteen femtomoles of 32P 3′-labeled OL26, OL26-1T, OL26-3T and OL26-6T was incubated for 15 min at 30°C with: lanes 1–4, 500 pmol of L193s + 11 pmol of htopoI; lane 5, 500 pmol of L193s; lane 6, 11 pmol of htopoI. (B) Quantification of three independent experiments as shown in (A).

Figure 4.

Figure 4

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htopoI-dependent recombination event. Autoradiogram of a 14% denaturing polyacrylamide gel. Fourteen femtomoles of 32P 3′-labeled OL26 was incubated for 60 min at 30°C with: lane 1, 5.5 pmol of htopoI; lane 2, 500 pmol of L193s; lane 3, 500 pmol of L193s + 2.2 pmol of htopoI; lane 4, 500 pmol of L193s + 4.4 pmol of htopoI; lane 5, 500 pmol of L193s + 8.8 pmol of htopoI; lane 6, 500 pmol of L193s + 11 pmol of htopoI. The mobility of the DNA fragments (nt) are indicated on the right-hand side.

Figure 7.

Figure 7

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The recombination reaction is stimulated by p53. (A) Autoradio gram of a 14% denaturing polyacrylamide gel. Fourteen femtomoles of 32P 3′-labeled OL26 was incubated for 30 min at 30°C with: lane 1, 50 pmol of L193s; lane 2, 50 pmol of L193s + 32 pmol of p53; lane 3, 50 pmol of L193s + 8.8 pmol of htopoI + 24 pmol of BSA; lane 4, 50 pmol of L193s + 8.8 pmol of htopoI + 16 pmol of p53; lane 5, 50 pmol of L193s + 8.8 pmol of htopoI + 32 pmol of p53. (B) Comparison of the stimulatory effect of p53 shown in (A) (average from three independent experiments).

RESULTS

Competition with the inactive htopoIY723F mutant

Previously, we have suggested that the htopoI double cleavage reaction may result in a release of the original htopoI cleavage complex (15). In order to further substantiate this hypothesis we used the suicide substrate L193s that carried a radioactive label at position 15 (Fig. 1) (15) and incubated it with wild-type htopoI in the absence or presence of the active site mutant htopoIY723F. The mutant htopoIY723F had no effect on the htopoI suicide cleavage (Fig. 2A, compare lane 3 with lanes 4–8). However, we found that the mutant enzyme inhibited the htopoI double cleavage reaction (Fig. 2B, compare lane 3 with lanes 4–8), whereas bovine serum albumin (BSA) showed no significant effect (Fig. 2B, compare lane 3 with lane 9, and see Fig. 2C). Up to 1000 ng of htopoIY723F inhibited the double cleavage reaction, whereas higher amounts of the mutant enzyme did not further change the cleavage efficiency. Hence, ∼40% of the htopoI double cleavages could not be reversed (Fig. 2C), most likely due to an inaccessibility of the 5′ OH group. Similarly, 40% of the htopoI double cleavages could not be reversed by high salt conditions (15). Both results can be explained by assuming a release of the suicide cleavage complex upon incision by the second htopoI molecule. The formation of such a gap makes a religation impossible.

Figure 1.

Figure 1

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The suicide substrate L193s. The htopoI cleavage site is indicated with an arrow. The radioactive label (32P) is located on nucleotide 15 counting from the 3′ end. See text for further details. BamHI restriction sites are indicated. Modified from Søe et al. (15).

Figure 2.

Figure 2

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The mutant Y723F htopoI can compete with the htopoI double cleavage. Wild-type htopoI was incubated with L193s and mutant htopoIY723F as described in the Material and Methods. (A) Autoradiogram of a 7.5% SDS–PAGE. Lane 1, no htopoI; lane 2, 500 ng of htopoIY723F; lane 3, 500 ng of htopoI; lanes 4–9, 500 ng of htopoI and 250, 500, 1000, 2000 and 4000 ng of htopoIY723F, and 4000 ng of BSA, respectively. (B) Autoradiogram of a 14% denaturing polyacrylamide gel. Load as in (A). A, b and c represent the htopoI double cleavages as previously shown in Søe et al. (15). (C) Comparison of double cleavage ‘A’ from three to five experiments as shown in (B).

Assay for measuring htopoI-induced recombination repair

Since the original cleavage complex is released upon incision by the second htopoI enzyme (as shown in Fig. 2), the double cleavage reaction may represent an initial repair event of the suicide cleavage complex. After such a release, a gap is formed that may serve as an entry site for a foreign DNA strand with homology to the gap. This foreign strand may then be ligated by the second htopoI to the 3′ end of DNA substrate in a recombination-like reaction. This in turn would lead to a complete repair of the original htopoI cleavage complex. Eukaryotic topoisomerase I has been shown to be able to perform recombination in vitro (17,18). Moreover, it has also been shown to be directly or indirectly involved in recombination events in vivo (19–22). In order to investigate if a recombination repair event takes place in vitro we developed an assay, which is shown in Figure 3. The non-labeled substrate L193s is incubated with wild-type htopoI and a suicide cleavage complex is formed. Subsequently, this complex is recognized by a second htopoI molecule, which cleaves upstream from the original suicide cleavage complex. Upon incision, the suicide htopoI cleavage complex is released and a 13 nt long gap is formed. A 26 nt long oligonucleotide with a 3′ label and a free 5′ OH group (OL26) is present in the reaction mixture. This oligonucleotide can hybridize to the predicted gap. The second htopoI molecule can now ligate OL26 to the substrate and a recombination repair event has taken place. The exact site of the recombination reaction can now be determined after incubation with BamHI, which cleaves the substrate at a specific position (27 nt upstream from the suicide cleavage site, see Fig. 1). The corresponding radioactive fragment is released and can be detected by electrophoresis through a sequence gel and subsequent autoradiography.

Figure 3.

Figure 3

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Experimental set-up for the investigation of a putative recombination-mediated repair pathway. The suicide substrate was incubated with htopoI whereafter both suicide cleavage and the htopoI double cleavage took place. See text for further details. The BamHI cleavage site was located 27 nt upstream from the htopoI suicide cleavage site.

htopoI-dependent recombination

Increasing concentrations of htopoI were incubated with non-labeled L193s in the presence of 3′ radioactively labeled OL26. Increasing concentrations of htopoI and subsequent cleavage by BamHI generated additional radioactively labeled products (Fig. 4, lanes 3–6). The BamHI cleavage site is located 27 nt upstream from the suicide cleavage site on the upper strand (see Fig. 1). The second htopoI cleavage was found to take place primarily 13 nt upstream from the suicide cleavage site (15). Therefore, if the second htopoI molecule would ligate OL26, a radioactively labeled oligonucleotide of 40 nt would be the predicted outcome after BamHI cleavage. In Figure 4 it can be seen that increasing concentrations of htopoI produced an increasing amount of a labeled fragment with a mobility of 40 nt (lanes 3–6). The appearance of this product was dependent on the presence of L193s (lane 1). This strongly supports that a htopoI-mediated recombination event had taken place (Fig. 4, compare lane 2 with lanes 3–6). Furthermore, it shows that the recombination event occurred at the predicted site of the second htopoI cleavage. However, additional reaction products also showed up with mobilities of 13, 14 and 16 nt, respectively (lanes 3–6). These can be explained as follows. When OL26 was ligated to L193s by the second htopoI this restored the primary suicide cleavage site and could be cleaved suicidally for a second time. Such a suicide cleavage would result in a product with a length of 14 nt (note that the label is at the 3′ end). With the htopoI preferential binding sequence the suicide cleavage can also take place 2 nt upstream from the primary cleavage site (17,23). In this case, a subsequent suicide cleavage results in a product with a mobility of 16 nt. The minor 13 nt long product most likely results from a suicide cleavage with a 5′ degraded OL26 with a length of 25 nt, which is also present in the reaction mixture (as it can be seen in Fig. 4, lanes 1–6 below the 26 and 40 nt long fragments). Therefore, all the four observed bands represent the same recombination repair event.

It could be argued that the observed recombination products did not arise from recombination events at the predicted position, but could have taken place elsewhere and that a different cleavage reaction by htopoI or a contaminating endonuclease resulted in the observed fragments. In order to rule out this possibility we investigated if the observed recombination products would be detected without BamHI cleavage. While all the previously mentioned recombination products can be detected after BamHI cleavage (with mobilities of 40, 16, 14 and 13 nt), omitting BamHI only gave rise to the recombination products with sizes of 16, 14 and 13 nt (Fig. 5, lane 3). This strongly supports the interpretation that the ligation step takes place 13 nt upstream from the suicide cleavage site and is catalyzed by the second htopoI molecule of the htopoI double cleavage complex.

The recombination event is dependent on correct base pairing

The alternative repair pathway for htopoI cleavage com plexes by a htopoI-dependent recombination-like reaction is predicted to require base pairing with a 13 nt long gap for an efficient repair event. To verify that a gap truly is formed upon the second htopoI cleavage and that correct base pairing with a complementary piece of DNA is required to seal the gap we used modified OL26 oligonucleotides that contained one, three or six mismatches at the 5′ end (OL26-1T, OL26-3T and OL26-6T, respectively) and investigated the recombination efficiency. As can be seen from Figure 6A, there is a strong dependency on correct base pairing of the incoming DNA strand (compare lane 1 with lanes 2–4). When the first base forms a mismatch the recombination efficiency is reduced to 40%. The efficiency is <10% with six mismatches present (Fig. 6B). However, as expected, the more mismatches present the more unspecific recombination events took place (Fig. 6A, lanes 2–4, marked with an asterisk). These products represent htopoI-mediated recombination events elsewhere on the DNA substrate where gaps can arise. However, it is important to distinguish these events from the recombination event that takes place at the double cleavage site and results in the products seen in Figure 6A (lane 1). Only this recombination event represents a specific repair of the htopoI cleavage complex.

The recombination reaction is stimulated by p53

Recently, we have found that p53 stimulates the htopoI double cleavage reaction (16). We were therefore interested in investigating if this stimulation also would influence htopoI-initiated DNA recombination-like repair. In lane 3 of Figure 7A the 40 nt long recombination product can be observed as well as the aforementioned 16 and 14 nt long recombination products. In the presence of a 2- or 4-fold molar excess of recombinant p53 over htopoI a strong stimulation of the recombination reaction was observed (Fig. 7A, compare lanes 4 and 5 with lane 3). Since p53 is a tetramer, this corresponds to a stoichiometry of 2:1 and 1:1 between htopoI and p53, respectively. It is worth noting that no 40 nt long recombination product can be detected in the presence of p53, but a strong increase in the 16 and 14 nt long recombination products (lanes 4 and 5). Since these products represent an additional suicide cleavage of the newly recombined substrate (as discussed above) this result shows that the cleavage activity of htopoI in general is stimulated by p53. Figure 7B shows that the maximum degree of stimulation by p53 is ∼6-fold.

DISCUSSION

In the present work we have shown that the htopoI double cleavage reaction results in the release of the original htopoI cleavage complex. The newly generated gap can serve as an entry site for a homologous DNA strand. The incoming strand is subsequently ligated by the second htopoI molecule that originates from the double cleavage reaction. Furthermore, we have shown that proper hybridization proximal to the cleavage site is important for an effective ligation reaction. Finally, we have demonstrated that p53 results in a strong increase of these ligation products.

In the past years it has been speculated that there may exist pathways for the repair of htopoI cleavage complexes. This was primarily based on the observations that cleavage complexes formed in the vicinity of various DNA lesions in vivo (10–12,24). A Tdp1 was identified in both yeast and humans (25–27). This enzyme is able to cleave the phosphodiester bond between the tyrosyl and the DNA backbone and thereby releases the cleavage complex. However, Pouliot et al. (25) also found that alternative repair pathways must exist for the removal of cleavage complexes. One possibility for such a repair pathway could be the here identified p53-stimulated htopoI-induced recombination-like repair (TIRR).

We have recently found that the tumor suppressor protein p53 stimulates the htopoI double cleavage in vitro (16). Furthermore, Mao et al. (24) have shown that htopoI double cleavages also take place in vivo in a p53-dependent manner after UV damage. Therefore, the double cleavage reaction may serve a certain purpose. We suggest the following scenario. The cell is damaged and the p53 protein level is upregulated. htopoI is activated by p53 and cleaves in the vicinity of a fraction of the DNA lesions. The induction of htopoI cleavage complexes may serve as a ‘sensor’ for the extent of damage. If too many complexes are formed the cell may go into apoptosis in a similar fashion as it is the case with CPT-induced complexes (reviewed in 28). Radford (29) suggested a similar role for htopoI in apoptosis induced by ionizing radiation. However, if only a few htopoI complexes have formed and signal that the damage level is low, these complexes need to be repaired. Here, TIRR may come into play together with the already identified Tdp1 pathway. The recombination repair pathway by htopoI has the advantage that it must not necessarily be dependent on the presence of double strands breaks and entry into the S-phase as it is the case for Tdp1 (30). As we have shown, a gap is formed after the release of the original htopoI cleavage complex and this can serve as an entry site for a DNA strand with sequence homology to the formed gap. This implies that such a repair event only needs micro-homology, perhaps for the price of being error-prone. It is also possible that the repair event takes place at a double-strand break and the second htopoI cleavage complex may then generate an overhang to which another strand with micro-homology hybridizes. The second htopoI could again serve as a DNA ligase.

Since both htopoI cleavage complexes and double cleavage complexes form at a DNA lesion in a p53-dependent manner in vivo (24) and we have found p53 to stimulate TIRR, it may suggest that the TIRR pathway described above functions in a p53-dependent manner. This in turn could be a part of the explanation for the observed genomic instability in cells expressing high levels of mutant p53 (31). A constantly activated p53 that has lost the transactivation function, but still can stimulate htopoI (32,33), would be expected to lead to increased recombination events and thereby contribute to genomic instability. This was recently described by El-Hizawi et al. (34). These authors found that gene amplification induced by CPT was greatly increased in cells expressing a ‘gain of function’ mutant of p53. These results suggest that htopoI cleavage complexes were involved in gene amplification. Moreover, this type of gene amplification was 10–25-fold increased when mutant p53 was expressed. Hence, p53-stimulated TIRR may at least partially explain these in vivo observations. Thus, TIRR may be an important pathway that can lead to genomic instability and cancer when it gets out of control.

Acknowledgments

ACKNOWLEDGEMENTS

We wish to thank Dr Karl-Heinz Gührs and Dr Tinna Stevnsner for fruitful discussions and suggestions during our experimental work. Furthermore, we wish to thank Dr Philippe Clertant for sending us the htopoIY723F-expressing baculovirus clone. This work was supported by the Deutsche Krebshilfe.

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