Recombination at chromosomal sequences involved in leukaemogenic rearrangements is differentially regulated by p53
Gisa S. Boehden1,2,
Anja Restle1,
Rolf Marschalek3,
Carol Stocking2 and
Lisa Wiesmüller1,2,4
1 Universitätsfrauenklinik, Prittwitzstrasse 43, D-89075 Ulm, 2 Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg and 3 Institut für Pharmazeutische Biologie, Johann Wolfgang Goethe Universität, Biozentrum, N230, R303, Marie-Curie-Strasse 9, D-60439 Frankfurt/Main, Germany
4 To whom correspondence should be addressed Email: lisa.wiesmueller{at}medizin.uni-ulm.de
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Abstract
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Chromosomal translocations and retroviral integration events at breakpoint cluster regions (bcrs) have been associated with leukaemias. To directly compare the effect of different cis-regulatory sequences on recombination, we adapted our SV40 based model system to the analysis of correspondingly selected bcrs from the TAL1, LMO2, retinoic acid receptor
(RAR
) and MLL genes. We show that a 399 bp fragment from the MLL bcr is sufficient to cause a 34-fold stimulation of spontaneously occurring DNA exchange and to respond to etoposide by up to 10-fold further elevated frequencies, i.e. to mimic the fragility of the 8.3 kb bcr during chemotherapy. To analyse the regulatory role of p53 in recombination involving leukaemia-related sequences, we stably expressed wtp53 and a transactivation negative mutant. Consistent with the proposed role of p53 as a suppressor of error-prone recombination, both p53 proteins down-regulated recombination with most of the sequences tested, even with the MLL bcr after etoposide treatment. Surprisingly, however, p53 stimulated recombination, in constructs carrying the RAR
bcr fragment. This is the first study, which provides evidence for a stimulatory role of p53 in homologous recombination. Our data further indicate that inhibition of topoisomerase I can mimic the effects of p53 on stimulating recombination on the RAR
bcr. Thus, these data also firstly describe a biological role of the biochemical interactions between p53 and topoisomerase I that may have implications for a gain-of-function phenotype of certain p53 mutants in genetic destabilization.
Abbreviations: bcrs, breakpoint cluster regions; BLM, Bloom syndrome protein; DSB, double-strand break; HR, homologous recombination; RAR
, retinoic acid receptor
; RGC, ribosomal gene cluster
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Introduction
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Chromosomal rearrangements, such as translocations, deletions and gene amplifications, initiate haematopoietic malignancies (1). Among the biochemical events potentially underlying these chromosomal instabilities, dysfunction of double-strand break (DSB) repair has closely been linked to tumorigenesis. The DSB repair pathway of non-homologous end-joining (NHEJ) reseals DSBs in mitotically growing cells and assembles variable regions during V(D)J recombination in developing lymphocytes. Homologous recombination (HR) enables genetic mixing during meiosis and repairs DSBs and other DNA lesions that remain unrepaired until being encountered by a replication fork (2). HR by gene conversion predominates among DSB-induced interchromosomal recombination events (3).
Molecular studies of genes involved in chromosome aberrations of leukaemia patients unveiled a striking clustering of translocation breakpoints, raising questions about the causes of local fragilities. In close to 30% of patients with T cell acute lymphoblastic leukaemia (T-ALL), alterations in the TAL1 (T-cell acute leukaemia 1) gene (also called SCL and TCL5), either as a consequence of a t(1;14) chromosome translocation or a deletion (4,5), are detected. The t(11;14) translocation, which involves the LMO2 (LIM domain only 2) gene (also called Rhombotin 2, Rbtn2 and TTG-2), is found in 510% of T-ALL cases (6). Acute promyelocytic leukaemia (APL) is, in the majority of patients, associated with a reciprocal translocation between chromosome 15 and chromosome 17, and the breakpoint on chromosome 17 lies within the retinoic acid receptor
(RAR
) locus (7,8). The MLL (mixed lineage leukaemia) gene (also called ALL-1, HRX and Htrx1) on chromosome 11 is rearranged in ALL and AML (9). So far, >30 hybrid genes of MLL with different fusion partners have been identified. In addition, MLL is a hotspot for chromosomal rearrangements after treatment with topoisomerase inhibitors (10,11).
Support for a direct involvement of p53 in HR processes came from the discovery of p53 mutants with separable functions in transcriptional transactivation, growth control and apoptosis induction versus HR (1215). Moreover, by use of an I-SceI meganuclease coupled DSB repair test system, an indirect effect stemming from an involvement of p53 in other DNA repair pathways was excluded (15). With respect to the underlying mechanism it is interesting that p53 recognizes mispaired recombination intermediates with high affinities in vitro and suppresses inappropriate recombination between mispaired DNA sequences and between sequences with short homologies in living cells (1517). The mismatch repair factor MSH2, which similarly recognizes mispairings in heteroduplex joints and restrains the exchange between divergent sequences, stimulates p53 binding to Holliday junctions and shows a common nuclear subcompartmentalization with p53 at sites of recombinative repair complexes (18,19). This has led to the proposal of complementary or even synergistic roles of p53 and MSH2 in the fidelity control of HR (16,19). Additionally, p53 was shown to interact with other surveillance factors of DSB repair, namely with BRCA1, which channels DSB repair into the non-mutagenic pathway of HR, with BRCA2, which promotes DSB repair via the conservative HR pathway, and with the anti-recombinogenic RecQ helicases, Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) (2025). p53 was further reported to counteract Holliday junction unwinding by BLM and WRN in vitro, suggesting that p53 regulates recombination indirectly by modulating other surveillance activities (24). However, cell based studies revealed that p53 and BLM act on complementary recombination regulatory pathways and indicated that p53BLM interactions may rather be required to recruit p53 to sites of aberrant DNA exchange processes (25). Finally, p53 was also demonstrated to bind HR proteins, namely Rad51, the initial strand transferase, and Rad54, a member of the SWI2/SNF2 family of helicase-like proteins (2528), and recombination down-regulation by p53 was found to depend on the Rad51 pathway (12,15,28). Co-localization of p53 with Rad51 and Rad54 was observed at putative processing sites of DNA breaks and at stalled replication forks, consistent with the observed involvement in the control of replication-associated recombination processes (29,30). In conclusion, current models on the anti-recombinogenic role of p53 involve physical interactions with the DNA intermediates of recombination and/or with Rad51 as the key events that are influenced by the presence of other proteins. The functional relevance of the association of p53 with recombination surveillance factors other than BLM awaits further clarification.
It is still difficult and time-consuming to quantitatively evaluate genetic alterations by conventional cytogenetics. Moreover, deletions or smaller rearrangements escape detection by this method. In order to elucidate cis-acting mechanisms for genomic instability, we introduced a set of leukaemia-related DNA fragments into our SV40 based assay (31) and assessed the impact of recombination regulation by p53 on element-specific rearrangements.
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Materials and methods
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DNA-cloning procedures
Retroviral integration sites from growth factor-independent mutants after retroviral infection of either the TF-1 or FDC-P1(M) cell lines (32,33) were isolated by either inverse-PCR or by cloning in lambda vectors. TF-1 mut 33 and FDC-P1(M) mut 2GM20 contained a single provirus in either the TAL1 or LMO2 gene locus at position 145233145232, GenBank accession number NT_032977, or 142546142547, genome clone RP23-358C5.
The Cla linker was introduced into XbaI/BamHI digested, Tag intron negative pUC-SV40-tsVP1(286S) (16) via PCR amplification between SV40 genome positions 2338 and 2682, and XbaI/BglII cloning. To generate further pUC-SV40-Cla vectors, we transferred the Cla linker after BamHI/ApaI or SfiI/XcmI cleavage. Next, we introduced PCR amplified fragments into the ClaI site. The
EGFP (15) fragment encompassed position 7421058 in pEGFP-N1 (Clontech, Palo Alto, CA), the TAL1 bcr 145372145111, accession number NT_032977, the LMO2 bcr 2531464025314212, accession number NT_009237, the RAR
bcr 9192091640, accession number AC090426, the MLL bcr 70366638, accession number X83604. Each vector set with a pUC-SV40-Cla, a pUC-SV40-tsVP1(290T)-Cla and a pUC-SV40-tsVP1 (196Y)-Cla derivative was sequenced and carried the foreign sequence in the same orientation.
Cell lines, virus, DNA synthesis and HR
CV1, COS1, LLC-MK2(p53her), LLC-MK2[p53(138V)her] and LLC-MK2(neo) cells were clonally established, cultivated and activated by 1 µM ß-estradiol (Sigma, Taufkirchen, FRG) as described (13,16). Virus particles were generated in COS1 cells and MOIs and PFUs determined as in ref. (31). The deletion of the foreign DNA insert during viral amplification was ruled out by restriction analysis of isolated viral DNAs. De novo synthesis of viral DNA was quantified as described (30). Briefly, cells on 60-mm plates were labelled with 30 µCi of [3H]thymidine at various times post-infection (hpi) for 1 h, viral genomes were isolated, and [3H]thymidine incorporation determined. Rates of viral DNA synthesis were expressed as counts per minute (c.p.m.) per 105 cells and mean values including SEs calculated. HR assays were executed and evaluated as before (16). The statistical significance of differences was determined using Student's unpaired t test.
Gel mobility shift assays
Fractions enriched for p53 were obtained after protein over-expression in insect cells and sequential nuclear extraction as described (13,16). 32P-Labelled DNA fragments were synthesized as in Wiesmüller et al. (31). The RAR
bcr fragment was amplified as described for cloning. The ribosomal gene cluster (RGC) repeat was amplified after SmaI/HincII subcloning from pSK-45-13-2-PyCAT (34) into the Cla linker of pUC-SV40-Cla by use of the primer pair 5'-AGGAGGAGATCTATCGATACCCGGGCAATTGT TGTTGTTAACTTGTTTATTG-3'/5'-GTTAACAACAACAATTGCCCC-3'. For purification of the radiolabelled fragments we used the QIAquick PCR purification kit (Qiagen, Hilden, FRG) according to the manufacturer's instructions, for the determination of DNA concentrations DNA Dipsticks (Invitrogen, Karlsruhe, FRG). Labelled DNA fragments (100 pM), competitor oligonucleotide (1000 nM) and 0.72.9 nM p53 proteins were mixed, incubated on ice for 30 min, electrophoresed on a native 4% polyacrylamide gel, and autoradiographed as in Dudenhöffer et al. (16).
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Results
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Assay to test cis-regulatory sequences in recombination
To study the influence of disease-related sequences on recombination, we utilized our assay based on genetic exchange between differently mutated SV40 minichromosomes (31). It relies on ts SV40 variants (SV40-tsVP1), which enabled us to produce virus particles at the permissive temperature of 32°C and to assay recombinative reconstitution of SV40-wtVP1 after co-infection with two SV40-tsVP1 mutants at the non-permissive temperature of 39°C (Figure 1A). To obtain HR frequencies, the ratios between the values from double infections with SV40-tsVP1 mutants and from control infections with the same infectious units of SV40-wtVP1 were determined. This procedure also served to exclude rate deviations caused by growth regulatory and cytotoxic effects, and alterations in virus propagation.

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Fig. 1. Test system for recombination at cis-regulatory DNA sequences. (a) Test principle. SV40 genomes were designed for insertion of foreign DNA by removal of the Tag intronic sequences and the introduction of a Cla linker and tsVP1 mutations. Virus particles were produced at the permissive temperature of 32°C and used for co-infection at the non-permissive temperature of 39°C, to select for SV40-wtVP1-Cla reconstitution by HR. SV40-wtVP1-Cla virus release was scored by plaque assays at 39°C. (b) Genomic organization. Squares represent exons; black lines introns (TAL1, GenBank accession number NT_032977, LMO2: NT_009237; RAR , AC090426; MLL, Z69744-Z69780). Coding sequences are marked with grey colour, untranslated sequences with grey stripes. tald deletion and t(1;14) translocation sites found in the TAL1 gene of T-ALL patients (4,5), bcrs in the LMO2 gene of T-ALL patients (6) and the RAR gene of APL patients (7,8) are indicated. The t(4;11) translocation sites, which show a clustering 5' to exon 12 of MLL in ALL patients below 1 year of age at diagnosis, are marked by black bars (35). Relevant features within PCR amplified bcr fragments are indicated (bottom).
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In order to permit packaging of additional DNA pieces of up to 0.7 kb, the SV40-tsVP1 genomes and the wtSV40 control chromosome were made smaller. This was achieved by removing 269 bp from the large tumour antigen (Tag) intron (Figure 1a). To allow the uptake of foreign DNA, we separated the early and late polyadenylation signals by a PCR strategy, which concomitantly inserted two new restriction sites (Cla linker).
Into the Cla linker of the corresponding SV40-wtVP1, SV40-tsVP1(196Y) and SV40-tsVP1(290T) genomes, we chose to introduce fragments of <0.5 kb that were derived from genomic bcrs for which frequent chromosomal breakage was expected from a clustering of deletions or translocations (Figure 1b). Thus, we PCR-amplified a 0.3 kb region within the RAR
bcr from APL patients, where two neighbouring t(15;17) translocation sites were identified at the molecular level (7,8). Additionally, we selected a 0.4 kb fragment from the MLL bcr, within which we have identified previously eight out of 24 and four out of 36 t(4;11) translocations in infants and other ALL patients, respectively (35). Furthermore, from the analysis of retroviral integration sites of factor-independent mutants generated by retroviral insertional mutagenesis of two myeloid progenitor cell lines (32), we found that two out of a total of 42 integration sites mapped to bcrs in ALL: one at the TAL1 locus associated with t(1;14) translocations (4,5) and one within the LMO2 gene, where t(11;14) translocations have been identified in patients (6) (Figure 1B). Since retroviral integration represents another type of genetic alteration related to chromosomal breakage, which is underlying the development of leukaemia (36), we also amplified the 0.3 and 0.4 kb regions encompassing the retroviral integration locus adjacent to the TAL1 and the LMO2 bcr, respectively. Finally, a 0.3 kb fragment from a mutated EGFP gene (
EGFP) (16), which has not been implicated in genome instability, served as a control.
Recombination is stimulated by the MLL bcr sequence
First, we measured HR in LLC-MK2(neo) cells for the wtSV40 virus set [SV40-tsVP1(290T), SV40-tsVP1(196Y) and wtSV40] and the SV40-Cla virus set, which demonstrated that the mutations created within the Cla linker genomes did not interfere with HR (Table I). Control viruses with the
EGFP sequence recombined at a frequency not significantly different from Cla linker viruses, indicating that changes in the total homology length (5439 versus 5137 bp) did not affect the HR frequency. The frequencies determined for TAL1, LMO2 and RAR
bcr viruses indicated a 2-fold stimulation in relation to the wtSV40 virus set, although compared with
EGFP virus the significance of the changes was limited (TAL1, P = 0.042; LMO2, P = 0.041; RAR
, P = 0.175). The MLL bcr fragment caused a 34-fold increase in the basal frequency (MLL, P = 0.002), i.e. maximal enhancement of spontaneous recombination among the cis-regulatory sequences investigated in this work.
Recombination regulation by p53 as a function of the DNA sequence
To test whether the non-viral sequences, influence the potential of wtp53 to regulate HR, we altered the p53-status in the SV40-infectable monkey cell line LLC-MK2, devoid of wtp53 (31). We newly established independent cell clones ectopically expressing the wtp53 hybrid protein p53her (data not shown), which is activated in a ß-estradiol-dependent fashion (13,16). To dissect functions in transcriptional transactivation and growth control versus HR, we also stably expressed the transactivation defective (14,15) mutant p53(138V)her in two independent cell clones.
In accord with previous SV40 based studies, wtp53 down-regulated HR (16,31). On average, the frequency for the wtSV40 virus set was reduced by 4-fold in four LLC-MK2(p53her) clones (2.6 x 104) as compared with LLC-MK2(neo) cells (Tables I and II). The frequency measured with Cla linker viruses was not significantly different than that found with the wtSV40 virus set. Similarly, the recombination frequencies observed after TAL1 bcr, LMO2 bcr and MLL bcr virus infections of LLC-MK2(neo) cells, were decreased by a factor of 23 in the LLC-MK2(p53her) clones (Table II). These findings indicate that wtp53 has the capacity to counteract DNA exchange events, even at MLL bcr sequences that cause a 34-fold stimulation of spontaneous recombination.
In contrast, with RAR
bcr virus, the frequencies were elevated 4-fold (P = 0.009) in a wtp53-dependent manner (Table II). Moreover, we found that in the two LLC-MK2[p53(138V)her] lines, the recombination frequencies were not significantly different from the frequencies in LLC-MK2(p53her) cells for wtSV40, Cla linker, TAL1 bcr, LMO2 bcr, MLL bcr and RAR
bcr virus infections. Clearly, both recombination inhibitory as well as stimulatory activities were exhibited by wtp53 and by mutant p53(138V). From this, both activities must be considered to be independent of the transcriptional transactivation functions of p53.
Role of cis-regulatory sequences in replication
HR is tightly linked to replication fork stalling and replication-associated HR processes were reported to be regulated by p53 (2,29,30). In order to study the impact of replication on recombination with respect to the selected DNA sequences, we measured [3H]thymidine incorporation into the viral genomes at different times after infection (hpi) (Figure 2). wtSV40 replication was described previously to remain either unaffected or to become slightly reduced by wtp53 (30,31). In LLC-MK2(neo) cells, similar replication patterns were seen with wtSV40, TAL1 bcr, LMO2 bcr and MLL bcr virus. Compared with wtSV40, replication of the RAR
bcr genome was increased 2-fold, 12 hpi, 24 hpi and 36 hpi. However, in LLC-MK2(p53her) cells no significant differences were found between the [3H]thymidine incorporations into wtSV40, TAL1 bcr, LMO2 bcr, RAR
bcr and MLL bcr virus genomes. Thus, the incorporation of the RAR
bcr sequence, which causes HR stimulation in a wtp53-dependent manner, did not alter viral replication in cells carrying wtp53.

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Fig. 2. De novo DNA synthesis. LLC-MK2(neo) (black rhombus) and LLC-MK2(p53her) (grey squares) cells were infected with the indicated virus strains and [3H]thymidine incorporation into viral DNA determined at various times post-infection (hpi). The maximum DNA synthesis rate for wtSV40 in LLC-MK2(neo) cells 24 hpi was taken as 100% (26 x 102 c.p.m./105 cells) and the relative rates calculated. Values are the means ± SEs from two to four independent measurements each.
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Analysis of RAR
bcr DNA in complex formation with p53
In an attempt to understand the stimulatory effect of p53 on HR between RAR
bcr virus genomes, we tested whether the RAR
bcr element represents a substrate for specific p53-interactions. For this purpose, we compared p53DNA complex formation by electrophoretic mobility shift assays with the 300 bp RAR
bcr fragment and with a 301 bp p53-specific binding sequence, namely the RGC repeat, which was characterized previously by Prives and colleagues (34). First, the RAR
and the RGC sequences were radioactively labelled by PCR amplification. Then, the DNA substrates were incubated together with protein fractions, highly enriched for baculovirally expressed human wtp53, the DNA contact mutant p53(248P), or the conformational mutant p53(273P) (13). After native gel electrophoresis of these mixtures, we detected p53RGC DNA complexes exclusively with wtp53, as indicated by the complete retardation of the labelled input DNA within distinct bands (Figure 3a). This picture did not emerge when we mixed the RAR
bcr probe with wtp53 at the same protein concentrations (Figure 3b). Thus, at 2.9 nM wtp53 caused the formation of p53DNA aggregates in the loading well, at lower concentrations (1.40.7 nM) the DNA was not retarded to a discrete position. Moreover, a similar smearing was observed with the p53 mutant proteins (Figure 3b, lanes 510). In summary, our data allowed us to draw the conclusion that p53 does not specifically and stably bind the RAR
bcr sequence, which stimulated recombination in a p53-dependent manner. This finding was consistent with the absence of a p53 consensus sequence within the RAR
bcr.
Sequence-specific recombination induction by topoisomerase inhibitors
p53 was reported to interact with topoisomerase II
, IIß and topoisomerase I that function in transcription, replication, recombination and DNA damage recognition (3741). To test the hypothesis that topoisomerase mediated cleavage within the RAR
bcr caused the p53-dependent increase of HR, we treated LLC-MK2(neo) and LLC-MK2(p53her) cells with the topoisomerase II inhibitor etoposide during recombination assays with RAR
bcr virus. For comparison, we analysed MLL bcr virus, because it has been proposed that topoisomerase II inhibition is involved in recombination associated with therapy-induced AML and ALL (42).
Etoposide exposure caused a 56-fold stimulation of recombination between MLL bcr virus chromosomes as compared with the 2-fold elevated DMSO values, which resulted in an overall increase by one order of magnitude (Figure 4a). Interestingly, LLC-MK2(p53her) cells were largely resistant to etoposide-induced recombination (0 µM, 7 x 104; 100 µM, 15 x 104), which indicated a 24-fold inhibition by wtp53. In contrast, neither recombination between wtSV40 nor between RAR
bcr viruses was affected by etoposide application, which excluded a major influence of topoisomerase II on the RAR
bcr sequence during recombination. When we administered the topoisomerase I inhibitor camptothecin to LLC-MK2(neo) cells, 34-fold elevated recombination frequencies with RAR
bcr virus (73 x 104 versus 23 x 104) were detectable (Figure 4b). After p53her expression, both DMSO and camptothecin treated cells recombined at similarly elevated frequencies (82 x 104 versus 64 x 104). Under the same conditions, camptothecin did not significantly influence recombination between wtSV40 or MLL bcr viruses. Taken together, the data suggest that topoisomerase I is responsible for p53 downstream events that might explain the striking finding that p53 stimulates recombination at the RAR
bcr fragment.
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Discussion
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In this work, a set of short sequences that are involved in leukaemogenic genome alterations was characterized with respect to their potential to regulate recombination in cis and the role of p53 in trans on this regulation. SV40 has been exploited previously as a powerful tool to examine the role of specific DNA structures (16). Here, we manipulated the virus in a way to enable us to quantitatively evaluate spontaneous and drug-induced recombination between chromatin-packaged circular SV40 genomes containing the sequence of interest.
Cis-acting mechanisms in leukaemogenic rearrangements
With respect to the individual sequences, we focused on loci where recurrent, non-random chromosomal translocations have been recognized in acute leukaemia patients. To narrow down the precise region, we combined two strategies: first, we took advantage of characterized breakpoint clusters available from patient data, and, secondly, we chose regions in which both chromosomal translocations and retroviral integrations were found.
Neither the TAL1 nor the LMO2 bcr fragment, which also corresponded to retroviral integration sites, caused a recognizable destabilizing effect in our assay, in contrast to the other two loci investigated. This may be due to the fact that sequences that dictate susceptibility to chromosome breaks were not included in the fragment examined or that the rhesus monkey kidney cells, in which these assays were performed, do not express the appropriate enzymatic machinery involved in this break. In this context it is important to note that both these bcrs are associated with T-cell ALLs and that the chromosomal joining occurred at the TCR
/
chain locus (46). Indeed, it has been demonstrated that heptamer/nonamer-like sequences within these loci function as weak V(D)J recombination signals (43,44).
Is it possible that the V(D)J recombination machinery also plays a role in the retroviral integrations characterized in this work? Interestingly, both the LMO2 and TAL1 integrations are found upstream (396 and 485 bp, respectively) of imperfect heptamer/nonamer-like sequences. Conceivably, misrecognition and DNA cleavage by the RAG complex and/or recruitment of DSB repair enzymes by RAG binding may have provided an entry site for the provirus, mediated by the viral-encoded integrase. Several recent reports have demonstrated the interplay between retroviral integration, and DNA repair, and thus lend support to such a hypothesis (45,46). At least one of the two cell lines in which these retroviral sites were identified also express RAG1 and RAG2 (M.Ziegler and C.Stocking, unpublished data). Further, V(D)J recombination was documented previously for a fraction of immortalized cell lines of the B-, T- and myeloid lineages (47). Retroviral integrations in the LMO2 locus have also been reported recently in the neoplastic clonal expansion of T-cells in two patients, following the successful gene therapeutic treatment of 11 children with severe combined immunodeficiency (SCID) (48). Although, integration into this locus has most probably influenced the selective growth of these cells, it cannot be presently excluded that preferential integration into this loci due to the V(D)J machinery contributes to the high incidence of this event. A better understanding of the potential interaction of V(D)J machinery and retroviral integration is thus required to assess the significance of these observations. As our assay was designed to identify cis-acting mechanisms in homology-directed recombinative repair rather than in V(D)J recombination, we can only conclude that the TAL1 and LMO2 regions tested did not influence HR in the absence of V(D)J recombinase.
In sharp contrast, we established recombination stimulation by the MLL bcr sequence in the SV40 based assay. This is the first report, which describes a cis-stimulatory role in recombination for a MLL sequence from the 8.3-kb bcr as small as 399 bp. Within this bcr two hotspots of chromosomal fusion sites were identified, one for infants below 1 year and one for patients above this age (35). Strikingly, within the first hotspot, which is covered by the MLL fragment chosen in this study, specific DNA cleavage, in response to treatment with topoisomerase II inhibitors, was described to occur (11,12). In agreement with these reports, we noticed a pronounced recombination induction after treatment with etoposide. Conversely, the MLL bcr was resistant to topoisomerase I inhibition under conditions, which caused a pronounced effect on recombination at the RAR
bcr. Surprisingly, the 399-bp MLL bcr fragment does not carry a topoisomerase II consensus site, although recognition sequences were identified at neighbouring positions between exon 11 and 14 (42). Alternative explanations for the MLL bcr fragility have come from observations indicating that it maps to the centre of a high affinity matrix attachment region (MAR), that MLL bcr subfragments still function as MARs, and that a correlation exists between the density of MLL translocation breakpoints and scaffold association (49). Topoisomerase II is enriched at MAR sequences, so that, after treatment with topoisomerase II inhibitors, the enzyme might preferentially cleave within the MLL bcr.
Recombination inhibition by p53 is sequence-independent
p53 was shown to be capable of inhibiting HR and NHEJ, and the recognition of aberrant exchange events may represent the underlying mechanism (1517). In agreement with these findings we saw down-regulation of ectopic recombination at the TAL1 bcr, LMO2 bcr and the MLL bcr in cell lines stably expressing wtp53 protein. Recombination frequencies were kept low, even when a dramatic rise of the exchange events at the MLL locus was provoked by etoposide treatment. Notably, rearrangements within the MLL bcr underly therapy-related leukaemias (42), and were observed to be closely related to mutations in the p53 gene (50). The consequence of this is that mutation of p53 increases the leukaemia risk, because of the failure to control DSB repair.
p53(138V) is defective in transcriptional activation and cell cycle regulatory activities. However, p53(138V) expressing cells were fully active in down-regulating ectopic recombination on TAL1 bcr, LMO2 bcr and MLL bcr virus genomes. These data further support the hypothesis that wtp53 directly controls recombination (1215). From our present knowledge there are three mechanisms possibly underlying recombination inhibition by p53 (1517,2428): first, by analogy to MSH2, it can be envisioned that p53 blocks continued strand exchange by the recombinase Rad51 after strongly binding to nascent intermediates of HR. Secondly, exonucleolytic proofreading of mispaired heteroduplexes may dissolve recombination junctions. Thirdly, p53 may act anti-recombinogenic via modulation of BLM activities, which disrupt recombinogenic molecules that arise at sites of defective processing of DNA replication intermediates.
Recombination stimulation by p53 is sequence-dependent
In sharp contrast to the data discussed so far, we saw a 4-fold p53-dependent recombination stimulation rather than an inhibition after introduction of the RAR
bcr fragment into the viral genome. It is well-established that replication fork pausing leads to elevated recombination activities (2). However, in LLC-MK2(p53her) cells, the replication curve for the RAR
bcr virus was indistinguishable from other curves such as the TAL1 bcr or LMO2 bcr-specific ones, suggesting that with respect to the stimulatory mechanism replication was not linked to recombination. Furthermore, the RAR
bcr fragment is missing a p53 consensus sequence and was not stably complexed by wtp53 in gel shift experiments. Thus, the recognition of the RAR
DNA sequence was not the initial cause of recombination enhancement by p53.
Notably, within the RAR
bcr fragment, two topoisomerase I recognition sequences (A/TGATG) are present (Figure 1B). When we applied camptothecin on the parental LLC-MK2(neo) line, we monitored a rise specifically in the RAR
bcr-dependent recombination frequency, as was expected for DNA breakage induced by topoisomerase I inhibition. However, in LLC-MK2(p53her) cells, we measured the same recombination frequency increase with and without camptothecin treatment. This indicated that the combination of camptothecin and wtp53 did not surpass the recombination-stimulatory effect by camptothecin alone, suggesting an epistatic pathway underlying recombination enhancement by topoisomerase I inhibiton and wtp53. In this context, we consider a recent report by Soe and colleagues (41), which describes that p53 stimulates the formation of a double cleavage complex containing two topoisomerase I molecules. This complex leaves behind a gap of
13 nt that supports strand exchange mediated by the second topoisomerase molecule in vitro. Thus, both camptothecin and p53 stabilize covalent DNAtopoisomerase I complexes and, therefore, may cause DNA breakage and recombination through the same enzyme.
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Conclusions
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Taken together, we have identified an 0.4 kb MLL fragment that markedly promotes ectopic recombination in cis. DNA recombination between MLL bcr viruses can be induced by topoisomerase II inhibition, which provides an experimental model for the chromosomal translocations leading to treatment-related ALL. The inhibitory effect of wtp53 directed towards these cancerogenic recombination events emphasizes the importance of p53 in restraining malignant progression by the surveillance of recombinative repair (15,16). In this work, we also discovered that p53 up-regulates recombination at a distinct sequence, namely at the RAR
bcr, and our data indicate mechanistic links to stable complex formation with DNA-bound topoisomerase I. Therefore, we speculate that p53 maintains genomic stability not only by controlling the homology length (15), but also by promoting homology-directed repair after the generation of certain types of DNA lesions and subsequent topoisomerase I cleavage complex formation (41). Damage-specific processing might also explain why Schiestl and co-workers (51) measured an increase of the recombination frequencies in p53+/+ but not in p53/ mice after X-ray treatment, whereas the benzo[a]pyrene-induced recombination stimulation was smaller in p53+/+ as compared with p53/ mice. Differential activation of p53 by distinct lesions may also underlie the fact that in vivo the HR-regulatory activity of p53 is developmentally regulated (52). Consistently, p53 is required for an irradiation-induced bypass pathway, to substitute for V(D)J recombination in scid mice, but counteracts spontaneous bypass rearrangements (53,54). Specific recruitment might coordinate the different repair-related activities of wtp53, as suggested by the tightly regulated interactions of wtp53 with topoisomerase I in response to DNA damage (39). However, both wtp53 and mutant p53 interact with topoisomerase I (38), and failure to suppress aberrant recombination coupled to the remaining capacity to stimulate DNA exchange would be expected to generate a gain-of-function phenotype in recombination, which could explain the rise in gene amplification observed with some mutant p53 proteins (55,56).
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Acknowledgments
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We are grateful to Dr Anne Dejean, Institut Pasteur, for the
phage containing the RAR
bcr locus, and to Prof. Dr B.Vogelstein, John Hopkins Oncology Center, Baltimore, for pSK-45-13-2-PyCAT. We thank Dr Christine Dudenhöffer and Evelyn Bendrat for help with mutant p53 and SV40 virus preparation. This work was supported by grants 10-1281-Wi and 10-1907-Wi 2 from the Deutsche Krebshilfe.
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References
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Received October 2, 2003;
revised December 22, 2003;
accepted January 14, 2004.