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Distinct Mechanisms of Nonhomologous End Joining in the Repair of Site-DirectedChromosomal Breaks with Noncomplementary and Complementary EndsAuthor(s): H. Willers, J. Husson, L. W. Lee, P. Hubbe, F. Gazemeier, S. N. Powell, and J. Dahm-DaphiSource: Radiation Research, 166(4):567-574. 2006.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR0524.1URL: http://www.bioone.org/doi/full/10.1667/RR0524.1

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RADIATION RESEARCH 166, 567–574 (2006)0033-7587/06 $15.00� 2006 by Radiation Research Society.All rights of reproduction in any form reserved.

Distinct Mechanisms of Nonhomologous End Joining in the Repair ofSite-Directed Chromosomal Breaks with Noncomplementary and

Complementary Ends

H. Willers,a,1 J. Husson,a L. W. Lee,a P. Hubbe,b F. Gazemeier,2 S. N. Powella and J. Dahm-Daphib,1

a Laboratory of Molecular & Cellular Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard MedicalSchool, Charlestown, Massachusetts 02129; and b Laboratory of Radiobiology and Experimental Radiation Oncology, Department of Radiotherapy

and Radiation Oncology, University Medical School Hamburg-Eppendorf, 20246 Hamburg, Germany

Willers, H., Husson, J., Lee, L. W., Hubbe, P., Gazemeirer,F., Powell, S. N. and Dahm-Daphi, J. Distinct Mechanisms ofNonhomologous End Joining in the Repair of Site-DirectedChromosomal Breaks with Noncomplementary and Comple-mentary Ends. Radiat. Res. 166, 567–574 (2006).

DNA double-strand breaks (DSBs) are considered the mostimportant type of DNA damage inflicted by ionizing radiation.The molecular mechanisms of DSB repair by nonhomologousend joining (NHEJ) have not been well studied in live mam-malian cells, due in part to the lack of suitable chromosomalrepair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysisof chromosomal NHEJ products in murine fibroblasts to focuson the error-prone rejoining of DSBs with noncomplementaryends, which may serve as a model for radiation damage re-pair. We found that noncomplementary ends were efficientlyrepaired using microhomologies of 1–2 nucleotides (nt) pres-ent in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2–3 nt). Micro-homology-mediated end joining was disrupted by Wortman-nin, a known inhibitor of DNA-PKcs. However, Wortmannindid not significantly impair the proficiency of end joining. Incontrast to noncomplementary ends, the rejoining of cohesiveends showed only a minor dependence on microhomologiesbut produced fivefold larger deletions than the repair of non-complementary ends. Together, these data suggest the pres-ence of several distinct NHEJ mechanisms in live cells, whichare characterized by the degree of sequence deletion and mi-crohomology use. Our NHEJ assay should prove a useful sys-tem to further elucidate the genetic determinants and molec-ular mechanisms of site-directed DSBs in living cells. � 2006

by Radiation Research Society

INTRODUCTION

Exposure of the cellular DNA to ionizing radiation in-flicts various types of damage. The DNA double-strand

1 Address for correspondence: Laboratory of Molecular & Cellular Ra-diation Biology, Department of Radiation Oncology, Massachusetts Gen-eral Hospital, Harvard Medical School, Charlestown, Massachusetts02129; e-mail: dahm@uke.uni-hamburg.de; hwillers@partners.org.

break (DSB) represents the principal lesion, which, if notadequately repaired, can lead to cell death through the gen-eration of chromosomal aberrations or the induction of ap-optosis. Alternatively, an inaccurately repaired DSB mayresult in mutations or genetic rearrangements in a survivingcell, which in turn can lead to genomic instability and car-cinogenesis. Complex damage response pathways haveevolved and are evolutionarily conserved to protect the cellfrom the potentially deleterious effects of a DSB. Two prin-cipal recombinational repair pathways have been recog-nized, homology-directed recombination and nonhomolo-gous end joining (NHEJ), which employ largely separateprotein complexes.

NHEJ involves the rejoining of double-stranded endswith no or little sequence homology. This can be an error-free or error-prone process and is determined in part by thetype of ends present. Complementary cohesive ends suchas typically generated by restriction endonucleases can bedirectly religated in an error-free manner. The repair of ex-ogenous DSBs induced by ionizing radiation is generallyerror-prone for two reasons: (1) Radiation destroys se-quence information by causing complex DNA damage sitesthat can span up to 20–30 bp (1, 2), and (2) the process ofNHEJ itself requires modification of the noncomplementarydouble-stranded ends prior to ligation.

The NHEJ pathway requires the heterodimeric Ku pro-tein, which binds to the ends of a DSB and recruits thecatalytic subunit of the DNA-dependent protein kinase(DNA-PKcs), thereby forming the DNA-PK holoenzyme.The active DNA-PK complex may recruit other factorssuch as the DNA ligase IV/XRCC4 complex. DNA-PK-mediated error-prone repair typically involves the align-ment of overhanging ends by pairing along nucleotides of1–4 bp microhomology that flank the break site, followedby gap filling by a DNA polymerase and/or trimming of afew bases, resulting in the ligation of ends with either theinsertion or deletion of a few base pairs. Cells defective inany of the core NHEJ proteins display radiation hypersen-sitivity due an impaired ability to close DSBs (3–5). Wort-

568 WILLERS ET AL.

FIG. 1. NHEJ substrate pPHW2. An artificial open reading frame(ORF) is defined by a new translational start sequence [Kozak (KOZ)sequence plus ATG], which is dominant over the gpt ORF. Simultaneouscleavage of both I-SceI recognition sites that closely flank the artificialKOZ/ATG site leads to loss or ‘‘pop-out’’ of the intervening sequence.NHEJ of the I-SceI break ends results in a reconstituted translation of thegpt ORF and subsequent resistance to XHATM in a colony formationassay.

mannin, a fungal metabolite, sensitizes cells to radiation,which seems to be mainly mediated through the inhibitionof the DNA-PKcs (6, 7).

The contribution of homologous recombination to DSBrepair has been studied extensively by employing plasmidsubstrates that integrate into the genome. The rare-cuttingI-SceI endonuclease has been used to induce DSBs at I-SceI recognition sites within a plasmid reporter gene [(8,9); for reviews, see (10, 11)]. In this system, homology-directed break repair leads to reconstitution of the reportergene. Compared to chromosomal homologous recombina-tion, the knowledge about intrachromosomal NHEJ in livemammalian cells is still limited due to the lack of selectablereporter systems, which are required to identify repairevents within a large cell population. Recently, severalstudies have used derivations of the I-SceI system to ana-lyze chromosomal NHEJ (12–15).

The induction of chromosomal DSBs by the I-SceI en-donuclease generates a 4-bp staggered cut that leaves 4-bpfree 3�-hydroxy overhangs, which are substrates for precisereligation. However, radiation creates far more complexchromosomal break ends than endonucleases. The technicalchallenge, therefore, is to design assays that employ endsresembling radiation-type damage more closely. To thisend, we previously designed and applied a plasmid-basedchromosomal recombination assay in which double-strand-ed ends that cannot be directly religated are produced 34nucleotides apart from each other (16). In the current study,we performed a physical analysis of a large number ofNHEJ products obtained with this assay and studied theeffect of Wortmannin in this process.

MATERIAL AND METHODS

Cell System

The cells and recombination substrates have been described previously(16). Mouse embryonic fibroblasts (MEF) expressing no or mutant Trp53carried a single chromosomal copy of pPHW1 (clone no. 24) or pPHW2(clone no. 35).

NHEJ Assay

Exponentially growing cells were either electroporated as described(16) or seeded at 5 � 105 in a T-25 flask and transfected the next daywith 5 �g pCMV-I-SceI or a control vector using Lipofectamine (Invi-trogen). To allow I-SceI expression and recombination to proceed, cellswere grown for 48 h in nonselective medium. In some experiments, Wort-mannin (Sigma-Aldrich) was added after I-SceI transfection at a finalconcentration of 20 �M. Control cells were mock-treated (DMSO, Sigma-Aldrich). Cells were then replated at appropriate densities between 103

and 105 per 10-cm dish and incubated for 18 days in XHATM (xanthine,hypoxanthine, aminopterin, thymidine and mycophenolic acid at 10, 13.6,0.17, 3.87 and 10 �g/ml, respectively; all from Sigma-Aldrich). NHEJfrequencies were calculated as reported (16).

Genomic DNA Analysis

For analysis of NHEJ products, XHATM-resistant clones were isolatedand expanded, and genomic DNA was extracted using the DNeasy tissuekit (Qiagen). The region containing both I-SceI recognition sites between

the SV40 promoter and the gpt gene was amplified by PCR using primersagctattccagaagtaggaggag and gtgatcgtagctggaaatacaaac. DNA sequencingof the amplified fragments was performed at the MGH DNA SequencingCore Facility or the Sequencing Service Laboratory of the University ofHamburg.

GFP Cotransfection Assay

The pCMV-I-SceI expression vector was cotransfected with a reporterplasmid expressing the green fluorescent protein (GFP) (pEGFP-N1,Clontech) at a 5 to 1 molar ratio. After 48 h, cells were subjected tosterile cell sorting (FACSCalibur, BD Bioscience). The top 5% of GFP-expressing cells were isolated, plated and grown in the absence ofXHATM. Genomic DNA was isolated as described above.

RESULTS

To study NHEJ of chromosomal breaks in mammaliancells, we used a recently developed plasmid assay that hasbeen described in detail elsewhere (16). Briefly, the plasmidsubstrate, pPHW2, contains an artificial translational startsequence inserted between an early SV40 promoter and thebacterial gpt gene (Fig. 1). The associated open readingframe (ORF) is shifted by 1 bp against the downstream gptORF and is dominant over the gpt start site, thus preventinggpt translation. Two I-SceI recognition sites flank the arti-ficial ATG site. Simultaneous cleavage at both I-SceI sitesresults in loss, i.e. pop-out, of the artificial ATG with re-constituted translation of the original gpt ORF, thereby al-lowing the detection of recombinants as colonies growingin XHATM selection medium. To model radiation-inducedDSBs, the tandem I-SceI sites are inverted, so that I-SceIcleavage results in the generation of noncomplementary 3�-OH single-stranded overhangs of 4 bases that are spaced

569NHEJ OF SITE-DIRECTED DSBs

FIG. 2. Repair product analysis after the generation of simultaneous I-SceI breaks in the pPHW2 substrate. Bold arrows indicate the I-SceI cleavagesites (bold type). Note that the single-stranded overhangs are not complementary because the I-SceI recognition sites are inverted. The two start codons(artificial and gpt) are italicized and underlined. Representative NHEJ products (a–f) are shown. Microhomologies in the overhangs used for end joiningare underlined. Inserted nucleotides at the break junction are in italics. The average number of deleted nucleotides at both sides of the breaks on thesense strand is shown. N, number of products; nt, nucleotides. Only repair products arising from the cleavage of both I-SceI are shown. Those eventsresulting from events at a single DSB are illustrated in Fig. 5.

FIG. 3. Effect of Wortmannin on NHEJ in cells containing pPHW2.The relative event frequency is plotted as a function of repair. Statisticalcomparisons were made using Fisher’s exact test (two-sided). Open bars:no Wortmannin; filled bars: Wortmannin.

34 bp apart and require sequence alteration prior to ligation(Fig. 2).

The pPHW2 substrate was stably integrated as a single-copy into the genome of immortalized mouse fibroblastswith nonfunctional Trp53 (16, 17). After transient transfec-tion of the I-SceI endonuclease, XHATM-resistant colonieswere expanded, genomic DNA was extracted and subjectedto PCR and sequencing analysis. A total of 64 clones wereisolated, of which 41 demonstrated the predicted pop-outevent with generation of noncomplementary ends and couldbe analyzed (Fig. 2). In 93% of cases, rejoining was me-diated by 1–2 nucleotides of microhomology. In almost allof these cases, A/T microhomologies present in the 3�-over-hangs were used (Examples a–c, Fig. 2), which was typi-

cally followed by ‘‘fill-in’’ synthesis of single-stranded gaps(Examples a and c). Accordingly, sequence deletion wasvery limited with a mean of 2.2 nt deleted prior to anneal-ing along microhomologies (range, 0–16 nt). There wereonly three products in which rejoining occurred indepen-dently of microhomologies, with a mean deletion size of3.0 nt (range, 1–4 nt). Perhaps surprisingly, we did notdetect any product that used microhomologies of more than2 nt even though these closely flanked the break sites, forexample GAT or CCTA (see Fig. 2).

NHEJ employing 1–4 bases of microhomologies hasbeen linked to the DNA-PK complex (5, 18). We thereforeasked whether the observed microhomology use could beimpaired by exposing I-SceI enzyme-transfected cells toWortmannin (at 20 �M), a known inhibitor of the DNA-PKcs. We found that treatment with Wortmannin resultedin a highly significant reduction of microhomology usefrom 93% to 62% (P � 0.011) (Fig. 3). However, the deg-radation of DNA ends was not increased: The mean dele-tion size was 2.8 nt (range, 0–10 nt) with Wortmannin com-pared to 2.2 nt in controls. There appeared to be a delay inthe closing of breaks, as suggested by an increased fre-quency of pop-out events and sequence captures upon in-hibition of DNA-PKcs (Fig. 3) (see the Discussion). Theobserved alterations in break rejoining in the presence ofWortmannin did not translate into a reduced cell survivalafter I-SceI break induction since the plating efficiencieswere similar in both Wortmannin- and mock-treated cells,62.2% and 59.8%, respectively (in two independent repeatexperiments). However, the fate of broken chromosomes interms of causing aberrations was not monitored. Consistentwith the cell survival results, the overall proficiency ofNHEJ as measured by the frequency of XHATM-resistant

570 WILLERS ET AL.

FIG. 4. Repair product analysis after the generation of simultaneous I-SceI breaks in the pPHW1 substrate, analogously to Fig. 2. pPHW1 generatescomplementary cohesive ends that are a substrate for direct religation.

colonies was at best only mildly reduced: 2.87 � 10�3 com-pared to 4.67 � 10�3 for Wortmannin- and mock-treatedcells, respectively. These data are in good agreement withour observation that NHEJ frequencies did not differ be-tween isogenic mouse embryo fibroblast strains either wild-type or null for Ku80 (Dahm-Daphi et al., unpublisheddata). The small amount of end degradation observed at theoverhangs (Fig. 2) shows that chromosomal DSBs withnoncomplementary ends are not subject to significant nu-clease attack, which has also been reported for extrachro-mosomal substrates (15).

We next asked whether chromosomal DSBs with com-plementary cohesive ends escape end degradation and areprecisely rejoined. We integrated pPHW1, which is identi-cal to pPHW2 except that the I-SceI sites are oriented as adirect repeat, into our fibroblast line (Fig. 4). We previouslyreported that the relative NHEJ efficiency was appreciablylower in clones carrying pPHW1 compared to pPHW2(mean, 1.3 � 10�3 compared to 1.15 � 10�2) (16). How-ever, extensive sequence analysis had not been performed.On further analysis, we found that only 54% of the iden-tified pop-out events resulted in precise religation of thecomplementary ends. In 46% of cases, there was end deg-radation, which in principle appeared to differ from theobserved rejoining of noncomplementary ends (Fig. 2).Compared to pPHW2, the proportion of microhomologyuse during rejoining was reduced to 38.5% (Fisher’s exacttest, P � 0.01). In addition, the deletion sizes increased byat least fivefold to a mean of 10.0 nt when NHEJ proceededalong microhomologies and to 23.1 nt without microhom-ologies, which was a statistically significant difference (ttest, P � 0.014).

When studying the effect of Wortmannin on cells car-rying pPHW1, we did not detect a reduction in cell survivalor NHEJ proficiency compared to mock-treated controls(data not shown), which was similar to the results withpPHW2. However, we were unable to sequence the corre-sponding repair products because the XHATM-resistantclones demonstrated instability at the chromosomal plasmidintegration site with likely amplification of the gpt se-quence. Repeated attempts to analyze frozen clones wereunsuccessful, yielding only mixed DNA sequences uponsequencing.

Therefore, as a second approach to study the fidelity ofrejoining of complementary ends, we analyzed the fate ofsingle-cut I-SceI sites in the pPHW2 substrate. CellularXHATM resistance could occur from a DSB generated atthe downstream I-SceI sites by three different mechanisms(Examples a–c, Fig. 5a): (a) End degradation extended up-stream into the artificial ATG site. (b) Small nucleotide de-letions (of 1, 4 or 7 nt) brought the artificial ATG site in-frame with the gpt start site (producing an N-terminal ex-tension of 13 amino acids). (c) Deletions of 2 or 5 nt gen-erated an adjacent downstream stop codon, therebyallowing translation at the gpt ATG. Other small deletionssuch as 6 or 9 nt are not detectable in our system becausethey do not change the reading frame. The repair resultsshown in Fig. 5b show that Wortmannin also reduced mi-crohomology use for the rejoining of complementary ends,although the numbers were too small to reach statisticalsignificance. Deletion sizes were difficult to interpret be-cause not all repair events led to XHATM resistance andthus to a selectable event.

Together, our results show that the extent of sequence

571NHEJ OF SITE-DIRECTED DSBs

FIG. 5. Analysis of repair products resulting from single cleavage at the downstream I-SceI recognition site incells carrying pPHW2. Panel A: Illustration analogous to Fig. 2. Panel B: Microhomology use and deletion sizewith and without Wortmannin exposure. The corresponding analysis of repair products resulting from double I-SceIcleavage is shown in Fig. 2.

FIG. 6. Repair product analysis of unselected clones (not grown in XHATM). Panel A: Flow cytometry-based cotransfection assay to isolate cellsthat exhibit high levels of GFP and I-SceI endonuclease. Panel B: NHEJ products from eight unselected clones.

deletion resulting from end processing during chromosomalDSB repair is limited, i.e., on average 2.3 nt for noncom-plementary ends (pPHW2) and 18.1 nt for complementaryends (pPHW1), with the largest deletion observed being 37nt. However, since the region between the viral promoter

and the gpt ORF comprises only approximately 100 bp,larger deletions could have been missed because theywould not have led to XHATM resistance. Therefore, wecotransfected the I-SceI expression vector with a GFP re-porter plasmid (Fig. 6a). The top 5% of GFP-expressing

572 WILLERS ET AL.

cells were sorted, plated and grown in the absence ofXHATM. This technique identifies those cells that containthe largest amount of transfected DNA and correspondinglythe highest levels of I-SceI endonuclease. Five out of sixrepair products had lost 0–8 nt, while only one clone con-tained a deletion large enough to destroy the adjacent gptgene (133 nt) (Fig. 6b). Thus, under both selective andunselective conditions, deletion sizes were limited. Of note,two clones (fourth and fifth row, Fig. 6b) showed end pro-cessing at both DSBs, providing evidence that cleavage andrepair at the two I-SceI sites occurred independently fromeach other.

DISCUSSION

Here we report the outcome of NHEJ of site-directedchromosomal breaks with noncomplementary or comple-mentary cohesive ends in murine fibroblasts. Noncomple-mentary ends were almost exclusively rejoined using 1–2nt of microhomologies present in the single-stranded over-hangs (Fig. 2). Lopez and colleagues recently reported onthe NHEJ of I-SceI DSBs in hamster CHO cells (14). Asin our system, these authors used I-SceI sites in inverseorientation to create breaks that required modification priorto ligation. They found that 55 out of 56 of deletion eventswere mediated by 1–2 nt of microhomology mostly presentin the overhangs. Together these two studies imply that therejoining of noncomplementary cohesive ends proceeds in-dependently of cell type and chromosomal location. Thesedata also suggest that radiation damage should be efficient-ly repaired if 1–2-nt microhomologies are present in over-hangs with 5� phosphate groups. However, radiation-in-duced DSBs often contain ‘‘dirty ends’’, i.e. phosphogly-colates or 3�-phosphate groups, which would be more dif-ficult to repair (19). Consistent with this idea, Pfeiffer andcolleagues showed in a cell-free system that rejoining ofradiation-induced DSBs was 10-fold less efficient and gen-erated significantly longer deletions (1–30 nt) compared toenzymatic breaks (average, 2.9 nt) but similarly relied on1–4-bp microhomologies at the junction (20). However,there is currently no in vivo system available that couldfaithfully model repair of site-specific radiation damage.

DSBs with complementary ends, which are principallysubstrates for precise religation, underwent error-prone re-joining at a high frequency, �50% (Fig. 4). This observa-tion compares well with other studies reporting error-pronerepair frequencies of 40–65% in CHO cells (14, 21). Incontrast to noncomplementary ends, the use of terminal mi-crohomologies seems to be reduced in this type of rejoin-ing, i.e. 34–68% (14, 15, this study). In this scenario, cellsappear to often use microhomologies distal to the end forstrand pairing combined with flap-end resection or mis-match correction. Alternatively, strands are resected suchthat blunt or abutted ends result, which are then ligated(22). Both repair modes may increase deletion size whencompared to a mechanism primarily employing microho-

mologies, which is consistent with our observations (amean deletion size of 23 nt compared to 10 nt for eventswithout and with microhomologies, respectively; Fig. 4).

Together, the data suggest the presence of at least fourprincipal NHEJ mechanisms, which can be operationallyclassified by the degree of sequence deletion and micro-homology use: (1) precise religation if ends are comple-mentary, (2) error-prone rejoining along microhomologiesflanking the break, (3) error-prone rejoining without use ofterminal microhomologies producing deletions that arelarger than with mechanism (2), and (4) rejoining of non-complementary ends that is minimally error-prone if 1–2 ntof microhomology is present in the overhangs. These repairprocesses are likely mediated by different protein complex-es.

To better understand the role of DNA-PKcs in the repairof one or two chromosomal DSBs, we employed Wortman-nin, a well-known kinase inhibitor (6). At the concentrationused (20 �M), Wortmannin was unlikely to interfere withother PIK-like kinases (23), although we cannot rule out aconcurrent partial inhibition of ATM. We found a signifi-cantly reduced microhomology use upon chemical inhibi-tion of DNA-PKcs (Fig. 2). In contrast, studies with variouscell-free, episomal and chromosomal assays have shownthat NHEJ-deficient cells preferentially use terminal micro-homologies for end joining (14, 18, 24–27). The existenceof an alternate NHEJ pathway that may use longer stretchesof microhomology has been suggested (26, 28). DNA-PKcsmay function as an end-bridging factor (29, 30) and trimDNA ends in concert with the Artemis nuclease (5). Toenable further or alternative repair steps to proceed, DNA-PKcs must dissociate from the DNA ends, which isachieved through autophosphorylation (31). Wortmannininhibits this autophosphorylation and may thus delay sub-sequent processing, such as polymerization and ligation(30, 32, 33) by other protein complexes such as LigIV/XRCC4. The physical presence of the inactive DNA-PKcsprotein may thus even suppress rather than promote the useof a microhomology-dependent pathway, which was ob-served when the NHEJ core proteins were absent (18, 24,27, 26, 34). A similar fundamental difference between ab-sence of DNA-PKcs and inactivation of its kinase activitywas previously suggested for the regulation of the balancebetween NHEJ and homologous recombination (13). Wort-mannin also caused an increase in the frequency of pop-out events and sequence captures (Fig. 3). These eventscould have been facilitated by a delay in the rejoining ofindividual I-SceI breaks due to inappropriate end bindingof inactive DNA-PK. Cleavage of both I-SceI sites mostlikely occurs independently of each other, as exemplified inFig. 6b. One DSB might have been repaired before thesecond DSB was induced. In most cases, however, bothDSBs were induced sequentially and remained open to gen-erate a pop-out event. It is also possible that two I-SceIendonuclease molecules can bind and cleave simultaneous-ly since the protein is small (28 kDa) and does not disso-

573NHEJ OF SITE-DIRECTED DSBs

ciate immediately from the DNA after cleavage (35). Sucha scenario would interfere with our interpretation of pop-out and single cleavage events. Although inhibition ofDNA-PKcs modified the repair process, we did not findevidence that the overall proficiency of repair was affected.By contrast, cells with compromised DNA-PK or LigIV/XRCC4 function are clearly defective in the rejoining ofradiation-induced DSBs. Since 1 Gy of radiation inducesabout 40 DSBs, we speculate that the cellular repair ma-chinery can compensate for the abrogation of DNA-PKfunction if the number of DSBs is small (i.e., one or twobreaks). However, with an increasing number of DSBs,such a compensatory mechanism may become inefficientor saturated. Alternatively, residual DNA-PKcs activity inthe presence of Wortmannin may account for the efficientrepair of a small number of site-directed DSBs.

In conclusion, plasmid-based chromosomal substrateshave proven useful to study NHEJ. However, since one ortwo site-directed enzymatic DSBs are unlikely to inducethe plethora of events triggered by radiation, these plasmidsystems may only partially model radiation-type damage.The evidence suggests that site-directed breaks may not re-produce all of the repair defects that can be seen after ir-radiation of Ku80- or DNA-PKcs-deficient cells. However,it seems likely that the loss of downstream effector proteins,i.e. XRCC4 or DNA ligase IV, would have a profound im-pact on the proficiency of I-SceI break rejoining; this willbe the subject of future studies.

ACKNOWLEDGMENT

This work was supported by a grant of the German Cancer Aid to JDD(Deutsche Krebshilfe grant No. Da-10-1510-2).

Received: December 24, 2005; accepted: May 9, 2006

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