Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53

Henning Willers1,2, Ellen E. McCarthy1, Petra Hubbe2, Jochen Dahm-Daphi2 and Simon N. Powell1,3

1 Laboratory of Molecular and Cellular Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA and
2 Laboratory of Experimental Radiation Oncology, Department of Radiation Oncology, University of Hamburg, 20246 Hamburg, Germany


    Abstract
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 Materials and methods
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We and others reported previously that the tumor suppressor p53 down-regulates spontaneous homologous recombination in chromosomally integrating plasmid substrates, but how p53 affects homology-dependent repair of DNA double-strand breaks has not been established. Furthermore, it has been hypothesized that p53 may suppress homologous recombination by direct interaction with recombination intermediates, but it is not known whether p53 directly acts on extrachromosomal plasmid substrates. In the present study, we asked whether p53 can suppress extrachromosomal spontaneous and double-strand break-induced homologous recombination. A plasmid shuttle assay was employed utilizing episomally replicating substrates, which carried mutated tandem repeats of a CAT reporter gene. Spontaneous homologous recombination and homology-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombination was found to proceed independently of the p53 status of isogenic mouse fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating single-strand annealing pathway, which is Rad51-independent, but also to Rad51-dependent gene conversion events. Comparison of homologous and non-homologous recombination frequencies revealed similar contributions to the repair of I-SceI-induced breaks irrespective of p53 status. Our results are consistent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. These data may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair in intact cells.

Abbreviations: DSB, double-strand break; HR, homologous recombination; IR, ionizing radiation; MEFs, mouse embryo fibroblasts; NHEJ, nonhomologous end-joining; SSA, single-strand annealing.


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 Introduction
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Homologous recombination (HR) processes must be tightly regulated to maintain genomic stability. Increased levels of HR are needed to ensure high fidelity repair of DNA double-strand breaks (DSBs), bypass of stalled replication or genetic transmission during meiosis. In contrast, spontaneous homology-dependent exchanges in mitotically growing cells must be considerably limited (1). Accordingly, the tumor suppressor gene p53 suppresses spontaneous inter- and intramolecular HR in several cell types, and this may be an important means by which p53 acts as a guardian of the genome (2–8). The mechanisms of this regulation are just beginning to be elucidated. In one report, p53 interacted with and blocked the activity of Rad51 recombinase, the central homologous pairing and strand exchange protein (9). Suppression of HR can be dissociated from p53's function at the G1/S checkpoint and even from its transactivation function (5,6,8).

It is not established whether the contribution of HR to DSB repair is suppressed in cells with functional wild-type p53. Homology-directed repair of chromosomal DSBs has been studied utilizing the rare-cutting site-specific I-SceI nuclease (10–16). From these studies, it has been concluded that repair via HR provides a significant alternative to illegitimate or non-homologous end-joining (NHEJ) and may account for up to 50% of all repair events. However, in most of the cell lines employed, p53 was commonly either mutated, as in the CHO lines (10–12,16), or not fully functional (14,15), as it seems to be the case in mouse embryonic stem cells (17,18). p53 has been linked indirectly and directly to NHEJ (19–22), but its precise function in this process remains to be defined. In response to DNA damage, p53 mediates G1 cell-cycle arrest, which is assumed to allow for adequate repair of damaged DNA before entering S-phase. Non-homologous end-joining is considered to dominate DSB repair in the G1 phase when no sister chromatid is available for homology-directed repair (20).

Recombinational mechanisms have been investigated using intra- as well as extrachromosomal substrates (5,7,16,23–25). Episomal DNA can replicate autonomously with the use of large T antigens, such as from the SV40 or polyoma virus, and replicating episomes associate with chromosomal chromatin (24,26). Extrachromosomal HR along close direct repeats is predominantly non-conservative with a resulting loss of sequence between the repeats, at least in murine cells (27,28). This mechanism can be explained by the single-strand annealing (SSA) model and does not depend on Rad51 function (16,28). In contrast, it has been reported that chromosomal HR involves in most cases conservative Rad51-dependent gene conversion, thereby maintaining the original sequence (4,11,13,28). p53 exhibits non-sequence-specific binding of DNA in vitro and in vivo (1,22). Importantly, p53 protein has been found to bind to artificial recombination intermediates in vitro and such an interaction could be required for suppression of recombinational exchanges (5). Consistent with this idea, Susse et al. (29) recently reported on the interaction of p53, Rad51 and heteroduplex joints in vitro. One hypothesis derived from those data is that the p53 protein should be capable of directly interfering with extrachromosomal recombination substrates in vivo, thereby suppressing HR.

In this study, we introduced episomally replicating plasmid substrates into mouse embryo fibroblast (MEF) lines that differed only in their p53 status. We hypothesized that p53-mediated suppression of HR would extend to extrachromosomal substrates, but may only apply to Rad51-dependent pathways. Furthermore, we sought to determine the relative contributions of HR and NHEJ to the repair of DSBs induced by the I-SceI nuclease with regard to p53 status.


    Materials and methods
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 Abstract
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 Materials and methods
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Cell culture, transfections and irradiation
Cell culture conditions and p53 status of the 10.1neo and 10.1/Val5 MEF lines have been described (8). For mammalian transfections, 3–4x106 cells at an incubation temperature of 31 or 37°C were suspended in 0.4 cm electroporation cuvettes with 800 µl PBS containing 20–40 µg circular recombination substrate and I-SceI expression vector, or a control. Electroporation was carried out at room temperature using a BioRad Gene Pulser at 960 µF and 0.28 kV. Electroporated cells were seeded immediately into 75 cm2 flasks containing fresh medium without selection antibiotics and were incubated for 24–96 h with media exchange every 24 h. For expression of p53 protein in wild-type conformation, 10.1/Val5 cells were switched from 37 to 31°C on the day before transfection. For experiments involving irradiation, a Siemens Stabilipan 2 X-ray generator was utilized (250 KVp, 12 mA, 2.08 Gy/min).

Plasmids
Plasmid pJH290 (30) was modified by removing the f1 Ig region (BglII fragment) and inserting a new AatII restriction site into the BglII site. The polyoma virus early region was removed as an AatII fragment from pJH290 and inserted into the unique AatII site of p#5CAT (23), generating the recombination substrate p#5CATpoly (Figure 1A and BGo). For construction of prCXi (Figure 3AGo), the polyoma early region was inserted into the AatII site of p5'->3'CAT (31), generating pRSV-CATpoly. The CAT gene was inactivated by insertion of an artificial I-SceI sequence into the unique BalI site, which was destroyed by this ligation. A homologous donor fragment of 520 bp, which lacks the 5' and 3' ends of the CAT gene, was created by PvuII and ScaI digest of p5'->3'CAT and inserted via blunt-end ligation into the unique HpaI site downstream of the I-SceI site-containing CAT gene. The recombination substrates were co-transfected with pCMV-I-SceI (23) or a control vector (11).



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Fig. 1. Assessment of non-conservative HR events in an extrachromosomally replicating substrate. (A) Plasmid substrate p#5CATpoly (9.9 kb) carries a tandem direct repeat of the CAT reporter gene. Deletions (3' and 5') render the CAT gene copies non-functional. Thus, there is an area of 0.4 kb of shared homology (black bars) within the genes. An I-SceI recognition site was placed between the repeats. The flanking sequences (RSV promoter and polyA sequence) have no function in the current assay. (B) In addition to the CAT genes, the plasmid contains the ß-lactamase (ampR) gene. The polyoma virus early region, which includes the origin of replication, enhancer element and coding sequence for T antigens, allows for stable episomal replication in mouse cells. (C) The fraction of replicated plasmid substrate was determined by the ratio of DpnI-digested to mock-digested aliquots from cells transfected with p#5CATpoly alone. Three cell lines were tested (Figures 1D and 2GoGo): 10.1neo at 37°C (filled diamond), 10.1neo at 31°C (open circle) and 10.1/Val5 at 31°C (open square). (D) Frequency of HR using p#5CATpoly in 10.1neo cells at an incubation temperature of 37°C plotted for spontaneous events and events induced by co-transfection of the I-SceI expressing vector. The logarithmic means with the range of independent duplicate experiments are shown.

 


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Fig. 3. Influence of p53 status on extrachromosomal spontaneous and break-induced gene conversion frequencies. (A) The episomal substrate prCXi (9.4 kb) is essentially analogous to p#5CATpoly except for the structure of the CAT gene repeats. The upstream gene copy was inactivated by insertion of a I-SceI site into the unique BalI site. A homologous donor sequence of 0.52 kb lacking the 5' and 3' ends of the gene was placed downstream in an inverted fashion (arrows indicate orientation of ORF). (B) 10.1neo and 10.1/Val5 cells were co-transfected with prCXi (10 µg) and pCMV-I-SceI, or a control (30 µg) and incubated at 31°C. Spontaneous and I-SceI-induced gene conversion frequencies were obtained at the 48 h time point. Non-homologous repair of the I-SceI induced breaks has no measurable contribution to the yield of camR colonies (data not shown). (C) Parallel to the studies shown in (B), these experiments were carried out at 37°C incubation temperature. At this temperature, 10.1/Val5 cells express a p53 protein that is in mutant conformation (p53-Val135). The lower detection limit in this experiment was 10–4. The logarithmic means with the range of independent duplicate experiments are shown.

 
Shuttle vector assay
The episomal shuttle assay was essentially carried out as described by Hesse et al. (26) and Lieber et al. (30). After various incubation times, transfected cells (1–3x106 per sample) were trypsinized, washed with PBS, suspended in 100 µl resuspension buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris–HCl pH 8.0 and 100 µg/ml RNAse) and incubated on ice with 200 µl chloroform and 200 µl lysis buffer (200 mM NaOH, 1% SDS) for 5 min. Following neutralization with 150 µl 3 M sodium acetate (pH 4.8), samples were centrifuged at 20 000 g for 5–10 min. Plasmid DNA was precipitated by transferring the top layer (400 µl) into 1000 µl ice-cold ethanol and incubating at <–20°C. The DNA was subjected to digest with DpnI restriction nuclease to remove plasmids with a bacterial methylation pattern (2 U enzyme for 2–10% of the sample), purified and dissolved in sterile ddH2O. A 10 µl aliquot of electrocompetent bacteria (DH10B; Gibco-BRL, Bethesda, MA) were electro-transformed with 10% of the replicated DNA in a total volume of 20 µl using 0.1 cm cuvettes (1.8 kV, 200 ). After a 1 h incubation in SOC (Gibco-BRL) medium, 100 µl aliquots directly from the transformed cultures or from serial dilutions were spread in parallel onto fresh LB agar plates containing 100 µg/ml ampicillin (amp) alone or containing ampicillin together with 20 µg/ml chloramphenicol (cam). Plates were incubated at 37°C for 16 h (amp plates) or 20 h (cam/amp plates). Plating efficiencies were determined by transformation of a control plasmid carrying intact amp and cam resistance (R) genes (pRSV-CATpoly) and plating onto cam/amp and amp plates. The plating efficiencies were found to amount to an average of 70%. Under the chosen conditions, 0.1 ng circular substrate DNA (9.4 or 9.9 kb) yielded in the order of 1x104 ampR colonies. Homologous recombination frequencies were based on the ratio of the total number of double camR and ampR colonies (containing recombined plasmid) to the total number of ampR colonies (containing all plasmid recovered) (26). Recombination frequencies originating from bacterial plasmid propagation were always determined in parallel. Those bacterial recombination frequencies were subtracted from the spontaneous and DSB-induced frequencies measured in the mammalian cells. Using the STBL2 strain of competent cells (Gibco-BRL), bacterial recombination for the p#5CATpoly and prCXi substrate amounted on average to 4x10–4 and 3x10–5, respectively. All frequencies were corrected for camR/ampR plating efficiency, which was determined for every individual transformation. Since absolute levels of recombination were found to vary between experiments, all comparisons were performed in parallel; hence, the relative differences, i.e. results of I-SceI expression versus none, p53 present versus absent or irradiation versus none, were highly reproducible among several independent repeats. Cell irradiations with 2 Gy, or higher doses (data not shown), were carried out 16 h after transfection, simultaneously with I-SceI expression and after the start of plasmid replication.


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Induction of I-SceI breaks yields homologous recombination frequencies of 5% in replicating plasmid substrates
To study HR in an extrachromosomal context, plasmid p#5CATpoly was generated (Figure 1A and BGo). In this plasmid, intra-molecular HR along direct repeats of the CAT reporter gene proceeds either spontaneously or can be triggered by an I-SceI-induced DSB which is placed between the repeats (Figure 1AGo). Homology-mediated reconstitution of the CAT gene is associated with loss of the intervening sequence and one repeat (23,31).

In principle, the recombination substrate with or without the I-SceI expressing vector was transiently transfected into MEFs. After incubation for various time intervals, the plasmids were extracted from the cells. All p#5CATpoly molecules that had not been fully replicated as well as the co-transfected I-SceI vector were eliminated by DpnI digest. Subsequent bacterial transformation yielded camR colonies, which contained recombinants generated by HR. The recombination frequency was calculated by the ratio of the total number of double-resistant camR and ampR colonies to the total number of ampR colonies correcting for plating efficiency and for contribution from bacterial recombination events. To verify the nature of the recombinants, camR colonies (n = 9) were isolated and plasmid DNA was extracted. Restriction fragment analysis indicated that all recombinants had lost one internal repeat and carried only a single functional CAT gene without any further plasmid rearrangement. Such a structure would be consistent with a SSA mechanism, which has been described to dominate extrachromosomal HR in murine cells (27,28). However, we cannot exclude the possibility that a crossing over mechanism gave rise to the products found.

Of note, depending on the particular bacterial strain used for plasmid propagation, a fraction of the spontaneously arisen recombinants recovered and analyzed was found to stem from recombination in bacteria (data not shown). Utilization of the STBL2 bacterial strain (Gibco-BRL) ensured that all possible events leading to camR colonies (bacterial and mammalian recombination) were consistent with SSA without any further plasmid rearrangements (see Materials and methods).

Figure 1CGo shows that after 48 h >50% of plasmids had undergone full replication as reported by Lieber et al. (30) using a similar system. The mean spontaneous HR frequencies in MEFs, that is in the absence of exogenous plasmid breaks, ranged from 2.5x10–3 to 6.5x10–3 at the 48 h time point. There was no significant further increase with longer incubation times (up to 96 h; data not shown). Figure 1DGo demonstrates that generation of DSBs by expression of I-SceI nuclease led to an ~7-fold increase in HR frequencies in two independent experiments. Thus, HR products could be found in up to 5% of all recovered plasmids.

Recombination consistent with the single-strand annealing model proceeds independently of p53
To determine the impact of p53 status on extrachromosomal HR, two MEF lines were employed. The p53-null 10.1neo line and the isogenic 10.1/Val5 clone, which expresses an exogenous temperature-sensitive p53 protein that is in wild-type conformation at <=32°C, have been characterized previously (8). Using the p#5CATpoly substrate, we found that at 31°C the p53-null cells displayed a mean HR frequency of 7x10–4 in the absence of I-SceI breaks (Figure 2AGo). The spontaneous frequencies in the presence of p53 were found to be virtually identical. For comparison, spontaneous HR frequencies previously obtained with the chromosomally integrating plasmid substrate pHWI in the same cell system are displayed (7,8). Briefly, pHWI carries two copies of the bacterial XGPRT gene each harboring a deletion mutation that inactivates gene function. Homologous recombination between these copies reconstitutes a functional gene that confers resistance to mycophenolic acid selection in a colony assay (see ref. 8 for details). In contrast to the episomal data, intrachromosomal HR was suppressed by p53 by more than one order of magnitude.



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Fig. 2. Influence of p53 status on spontaneous and break-induced extrachromosomal homologous recombination (HR) in comparison to chromosomal HR. (A) Extrachromosomal HR frequencies using the episomal substrate p#5CATpoly were assessed in 10.1neo cells (vector alone) and 10.1/Val5 cells [exogenous wild-type (wt) p53] at 31°C incubation temperature. Recombinants were recovered after a 48 h incubation period. For comparison, intrachromosomal HR frequencies are displayed, which were observed in 10.1 cells (p53-null) and primary MEFs (endogenous wild-type p53) carrying the chromosomally integrated substrate pHWI. It was previously found that the main determinant of HR frequencies when comparing primary and immortalized MEFs is the cellular p53 status (7). Homologous recombination frequencies were calculated based on the estimated HR rate per cell generation (taken from ref. 8) and the number of cell divisions per 48 h time period to facilitate comparison with the episomal HR frequencies. The logarithmic means with SEM for three to four independent experiments are shown. (B) 10.1neo and 10.1/Val5 cells were co-transfected with p#5CATpoly and pCMV-I-SceI or a control (10 µg each) and incubated at 31°C. Spontaneous and I-SceI-induced HR frequencies were obtained at the 48 h time point. The logarithmic means with SEM for three independent experiments are shown.

 
Co-transfection of the I-SceI expression vector and the HR substrate into 10.1neo cells led to a 32-fold increase in extrachromosomal HR frequencies above the spontaneous background (Figure 2BGo). The same elevation (28-fold) was seen in cells expressing p53. There was also no difference detected with incubation times of >48 h (data not shown). It was noted that the induction of HR frequencies following I-SceI expression was higher at 31°C than at 37°C, i.e. approximately 32- versus 7-fold. This was mainly due to a consistent 5-fold lower frequency of spontaneous HR events at 31°C, which presumably was a result of the reduced cell proliferation at that temperature.

The data in Figure 2AGo involve not only the comparison of extra- versus intrachromosomal recombination, but also the comparison of two genetically different homologous pathways. The episomal p#5CATpoly substrate allows scoring of events compatible with Rad51-independent SSA, and the chromosomal pHWI substrate measures Rad51-dependent HR due to the inverted repeat arrangement of the reporter genes, which precludes the occurrence of SSA (see below). Therefore, it was unknown whether the observed lack of p53 effect was due to the difference of intra- versus extrachromosomal per se or due to the independence of SSA from Rad51 function.

Extrachromosomal gene conversion proceeds independently of p53
Next, a plasmid substrate was employed which specifically allowed the scoring of Rad51-mediated gene conversion events (Figure 3AGo). This plasmid, designated prCXi, contains an upstream CAT gene copy inactivated by an I-SceI recognition site insertion. The downstream copy is mutated by 5' and 3' deletions and is oriented as an inverted tandem repeat. Hence, gene conversion events without crossing-over that lead to the repair of the I-SceI break can be directly selected for, whereas other events cannot yield camR colonies. Importantly, SSA events cannot occur due to the inverse orientation of the repeats; exonucleolytic processing of the ends would expose identical but not complementary single-stranded DNA tails, which are required for SSA. Individual camR recombinants (n = 10) were subjected to BalI digest and the diagnostic 1.1 kb band, which is indicative of gene conversion leading to replacement of the I-SceI site by BalI, was seen in all samples (Figure 3AGo and data not shown).

Gene conversion frequencies were stimulated 12–51-fold after DSB induction (Figure 3BGo). Again, there was no difference between cells null for p53 and those expressing the protein, neither for spontaneous nor for break-induced gene conversion frequencies. It has been hypothesized that p53 may exert its suppressive function in its non-induced, transcriptionally inactive state (5). Therefore, we tested whether the p53-Val135 mutant, which lacks G1/S checkpoint control capability and transactivation activity but which has retained the ability to suppress chromosomal HR (8), could affect gene conversion. However, analogously to wild-type p53, no suppressive effect was observed (Figure 3CGo).

It was noted that the absolute levels of repair by gene conversion were by one order of magnitude lower than the observed frequencies of SSA (Figures 2B and 3GoGo). Furthermore, when we used a prCXi derivative, designated prCXd, which carried the homologous donor in a direct repeat orientation, no increased yield of camR could be recovered after break induction (data not shown). Because the downstream homologous CAT donor copy in prCXd carries a 5' as well as a 3' deletion mutation (compare with Figure 3AGo), only repair via gene conversion can lead to chloramphenicol resistance. In contrast, repair of the I-SceI break via a SSA-type mechanism can occur but cannot result in chloramphenicol resistance. These data are consistent with SSA being the predominant extrachromosomal repair mechanism, at least with the gene arrangements chosen in the current study.

Similar contributions of homologous and non-homologous recombination to the repair of I-SceI breaks
To investigate the contribution of both HR and NHEJ to the repair of I-SceI breaks, the following approach was taken. Plasmid substrates rescued from cells transfected with p#5CATpoly and pCMV-I-SceI were subjected to I-SceI nuclease digest, thereby cutting all molecules that had not received any break or that had just undergone precise religation of the enzymatic break. Following bacterial transformation, ampR colonies were isolated and the structure of repair products was analyzed by restriction mapping.

Figure 4AGo displays the classes of recombinant molecules identified. Presence of SSA products was diagnosed by the demonstration of a new 2.2 kb HindIII fragment instead of the parental 2.6 kb band, which contains the non-functional CAT tandem repeat (Figure 4AGo). As predicted, these recombinants gave rise to camR colonies upon repeat bacterial transformation. The SSA products comprised 39–51% of all repair events occurring in non-irradiated cells. Non-homologous recombination products were defined by the loss of the I-SceI site, but without reconstitution of the CAT gene. These recombinants could be divided into two classes: (i) illegitimate rejoining of ends (`NHEJ'), introducing minor sequence alterations with preservation of the parental diagnostic HindIII fragment of 2.6 kb, and (ii) recombinational repair involving large deletions that originated from the break site, with a mean deletion size of 2 kb, thereby removing the upstream HindIII site (Figure 4AGo). The relative distribution of these repair types was not significantly different between cells with and without p53, irrespective of the presence of an additional DNA damage signal (IR) (Figure 4AGo).



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Fig. 4. Analysis of all repair events with respect to p53 status. (A) Illustration of classes of recombinants recovered from mouse embryo fibroblasts transfected with p#5CATpoly and pCMV-I-SceI after DpnI and I-SceI digest. Restriction fragment analysis using HindIII and others (data not shown) yielded diagnostic bands of the lengths indicated. CAS, sensitive to double-selection with cam and amp; CAR, resistant to double-selection. For each experimental arm, 31–45 ampR colonies were isolated and plasmid products that were resistant to I-SceI digest were analysed. In three cases, the product could not be classified and in one case, there was a complex recombinational rearrangement leading to a camR colony. Wt, wild-type; n, number of ampR colonies; ND, not determined; 0, no irradiation; IR, irradiation with 2 Gy. (B) Frequencies of homologous and non-homologous (NHEJ and deletional) repair events plotted for cells with and without irradiation and with and without presence of p53. Relative induction of repair frequencies with IR is displayed.

 
Finally, we asked whether exposure to IR was sufficient to increase absolute recombination frequencies, as seen in other experimental systems (28). We found an ~2-fold higher yield of camR colonies from the irradiated MEFs than from the unirradiated lines (Figure 4BGo and data not shown). However, this increase in homology-mediated repair was similar in p53-null and p53-expressing MEFs. The absolute non-homologous recombination frequencies, including `NHEJ' and `deletional' events, were indirectly estimated based on the observed ratio of homologous and non-homologous repair events among ampR colonies (Figure 4AGo) and the HR frequencies obtained directly from cam/amp plating. The largest IR-triggered enhancement of 4.5-fold was seen for illegitimate rejoining in MEFs with p53, which compared with a 2.2-fold enhancement in p53-null cells, but this difference was not statistically significant (P > 0.05).


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We report here that neither spontaneous nor DSB-induced HR in episomal plasmid substrates was affected by the p53 status of isogenic MEFs. This lack of p53 effect was observed for both Rad51-dependent gene conversion and Rad51-independent SSA events. In contrast to the extrachromosomal data, Rad51-dependent intrachromosomal HR was suppressed by p53 >10-fold in the same cell system (8). These results suggest that regulation of HR by p53 is specific to the high order structure of chromosomal chromatin. However, although p53 function has clearly been linked to chromosomal structure (32–34), further study is needed to obtain a testable hypothesis of how p53 specifically acts on chromatin-bound recombination substrates in vivo. Interestingly, Wiesmüller et al. (3) observed a significant suppressive effect of p53 on intermolecular HR between mutated SV40 genomes. In that assay, the SV40 substrates remain chromatin-bound through the entire course of infection, but do not integrate into the host genome. It is conceivable that there are functionally relevant differences in the chromatin association of SV40 genomes and replicating plasmid substrates.

The lack of p53 effect in the present study applied to wild-type protein as well as the Val135 mutant. Since p53-Val135 has been shown to be a suppressor of chromosomal HR (8), this finding rules out any bias that could have arisen from cell toxicity mediated by the wild-type protein. It also implies that the particular cell-cycle phase in which the cell population is positioned at the time of transfection, i.e. mostly G1 for wild-type cells and S/G2 for mutant or p53-null cells (8), does not significantly impact on intra-molecular HR or NHEJ.

The recombination substrates replicated due to the presence of the polyoma virus early region. The polyoma T antigen is not thought to interfere with any functions of p53 in recombination: in contrast to the SV40 virus, the polyoma T antigen is not known to interact with p53 (35), its removal from the recombination substrate did not reverse the lack of p53 effect (data not shown), and data from Wiesmüller et al. (3) suggest that suppression of HR by p53 is not dependent on DNA replication.

Using an episomal plasmid reactivation assay, we previously reported that p53 enhances NHEJ of cohesive break-ends following exposure to IR, presumably via the single-strand annealing function of the basic C-terminal end (22). Of note, since only linear plasmid was transfected, no homologous repair template was available. However, in the present study, which involved the repair of breaks by competing homologous and non-homologous pathways in replicating substrates, there was an IR-induced enhancement not only in the p53 wild-type, but also in the p53-null cells (Figure 4BGo). In contrast, in other studies reduced NHEJ with loss of wild-type p53 function was found (36,37). These observations imply that the cellular context or even the assay system used are important determinants of outcome, and they clearly contrast the universal suppressive effect of p53 on chromosomal HR, which has been observed in a variety of cell lines and experimental systems (2–8).

Interestingly, the function of BRCA2, which acts as a HR promoting protein together with Rad51, seems to extend to both, intra- and extrachromosomal substrates (F.Xia, D.G.Taghian, J.S.DeFrank, H.Willers and S.N.Powell, in preparation). In contrast, the effect of p53 on HR is limited to substrate DNA that is integrated into higher order chromatin structures. This finding is consistent with the idea that p53 functions at a regulatory level, which is upstream of the direct interaction with naked recombination intermediates. Our results, therefore, may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair. Ultimately, chromosomal plasmid assays need to be devised that resemble IR induced damage sites more closely. It is tempting to speculate that the role of p53 may be altered dependent on whether IR is used to assess a specific in vivo chromatin effect versus any observations made with artificial chromosomal repair substrates alone.


    Notes
 
3 To whom correspondence should be addressed Email: snpowell{at}partners.org Back


    Acknowledgments
 
We would like to thank Drs J.Hesse, M.Jasin, A.Levine and J.Nickoloff for their generous contributions of cells and plasmids, and Dr L.Wiesmüller, Dr H.Liber and the members of our groups for stimulating discussions and critical review of the manuscript. The technical assistance of Tim Phalen is gratefully acknowledged. H.W. was supported by a scholarship grant from the Dr Mildred Scheel Foundation for Cancer Research (Deutsche Krebshilfe). Part of this work was supported by a grant from the NIH to S.N.P. and a grant from the Deutsche Krebshilfe to J.D.D.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 27, 2001; revised July 6, 2001; accepted July 12, 2001.