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[Cancer Research 61, 2649-2655, March 15, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

Severe Combined Immunodeficient Cells Expressing Mutant hRAD54 Exhibit a Marked DNA Double-Strand Break Repair and Error-prone Chromosome Repair Defect1

Janice M. Pluth2,,3, Laura M. Fried2 and Cordula U. Kirchgessner4

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305-5152


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA double-strand breaks (DSBs) can be induced by a number of endogenous and exogenous agents and are lethal events if left unrepaired. DNA DSBs can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). In mammals and higher eukaryotes, NHEJ is thought to be the primary pathway for repair, but the role for each pathway in DNA DSB repair has not been fully elucidated. To define the relative contributions of HR and NHEJ in mammalian DNA DSB repair, cells defective in both pathways were produced. Double-mutant cells were created by expressing a dominant mutant hRAD54 protein in a DNA-dependent protein kinase (DNA-PK)-deficient severe combined immunodeficient cell line. Double-mutant cells demonstrate an increase in ionizing radiation sensitivity and a decrease in DNA DSB repair as compared with either single mutant, whereas single-mutant hRAD54 cells exhibit a wild-type phenotype. Unexpectedly, DNA-PK-null cells were more resistant to mitomycin-C damage than were wild-type cells. Chromosome aberration analysis reveals numerous incomplete chromatid exchange aberrations in the majority of the double-mutant cells after ionizing radiation exposure. Our findings confirm a role for HR in DSB repair in higher eukaryotes, yet indicate that its role is not evident unless the primary repair pathway, NHEJ, is nonfunctional. Mitomycin-C resistance in DNA-PK-null cells compared with wild-type cells suggests that the HR pathway may be more efficient in cross-link repair in the absence of NHEJ. Lastly, the incorrectly repaired chromatid damage observed in double-mutant cells may result from failed recombination or another error-prone repair process that is apparent in the absence of the two primary repair pathways.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA DSB5 repair is critical for the maintenance of genetic integrity. DSBs may occur spontaneously or be induced by IR or radiomimetic drugs. Inaccurate repair of DSBs may result in genetic instability and lead ultimately to carcinogenesis through the activation of oncogenes, inactivation of tumor suppressor genes, or loss of heterozygosity. There are two major DSB repair pathways, NHEJ and HR, and the relative contribution of each of these repair pathways in mammalian DSB repair is still being elucidated.

HR, as studied in yeast systems, uses a large region of homologous sequence, such as a sister chromatid or a homologous chromosome, as a template to restore a double-stranded break in DNA with high fidelity (1 , 2) . Saccharomyces cerevisiae RAD52 epistasis group proteins have been shown to be involved in HR with mutants displaying sensitivity to IR and chemicals known to induce DSBs (3, 4, 5, 6) . The most sensitive yeast phenotypes are the RAD51, RAD52, and RAD54 mutants, and mammalian homologues of these genes have all been identified and cloned (5 , 7, 8, 9, 10) . The role of these RAD52 epistasis group proteins in mammalian cells has been difficult to study, because knockout mice were either embryonically lethal (RAD51-/-) or displayed no apparent defect (RAD52-/-; Refs. 11, 12, 13 ). Essers et al. (14) reported that ES cells from RAD54-/- mice show reduced HR and increased IR sensitivity. Increased IR sensitivity was also observed in RAD54-/- chicken DT40 cells and RAD54-/- Drosophila (15 , 16) . Recently Essers et al., (17) further modified our understanding of the role of HR in repair of IR damage, by revealing that RAD54-deficient mice exhibit IR sensitivity only at an embryonic stage and not as adults.

The NHEJ pathway of DNA DSB repair does not rely on large regions of DNA sequence homology and often results in deletions and other genomic alterations; thus, it is a low-fidelity type of repair (18 , 19) . The three subunits of DNA-PK (Ku p70, Ku p86, and DNA-PKcs), the XRCC4 gene product, and DNA ligase IV, are all involved in NHEJ, and cells defective in any of these proteins are radiosensitive (20, 21, 22, 23, 24) . In yeast, HR is the primary pathway for DNA DSB repair; however, in mammals, NHEJ appears to be the major pathway (25) . Although HR is the dominant mode of DNA DSB repair in S. cerevisiae, NHEJ has been detected in yeast when HR is defective (26 , 27) .

Mouse SCID cells defective in DNA-PKcs are ~2- to 3-fold more sensitive to IR than its wild-type, parental strain, C.B-17 (28, 29, 30, 31) . This IR sensitivity is attributable to a defect in the SCID cells’ ability to perform NHEJ (29 , 32, 33, 34, 35) . The SCID cells’ defect in NHEJ results from a point mutation in the gene encoding for the catalytic subunit of DNA-PK (21 , 36, 37, 38) . Although a truncated protein is produced in SCID cells, it likely has little effect because the phenotype of DNA-PKcs-null mouse cells is essentially identical to that of SCID cells (39, 40, 41) .

Because NHEJ-defective SCID cells still manage to repair about one-half of DNA DSBs after IR (35) , we investigated whether the level of DNA DSB repair observed in SCID cells is attributable, at least in part, to the presence of the HR repair pathway. Others have also attempted to uncover the relative roles of HR and NHEJ in IR sensitivity and DSB repair. Recent work in mammalian cells using a tandemly repeated substrate integrated on a chromosome inferred the use of HR in up to 40% of all of the joining events, of which 30% were a deletion-type HR, and only 10% were gene conversion (42) . Given that HR uses homologous DNA as a template, there is probably a cell cycle preference to which pathway is used at any given time. HR is likely preferred during late S and G2 phases, whereas in other phases of the cell cycle, NHEJ is favored. This cell cycle-related preference for each of the pathways was recently documented in chicken DT40 cells (15) , with the authors concluding that both pathways contribute significantly to maintaining chromosomal DNA during the cell cycle. In addition, work using Drosophila KU70-/-/RAD54-/- double-mutants has shown that there is a synergistic effect of the two DSB repair pathways in repair of IR but not MMS damage (16) . Lastly, Essers et al. (17) have revealed that both pathways play a role in IR damage repair at different times during development using SCID/RAD54-/- knockout mice, with HR prominent during early development and NHEJ being the primary pathway in the adult animal.

In the present study, using a dominant-negative system, we show the effects of a double defect in HR and NHEJ on DNA DSB repair and chromosome repair in differentiated mammalian cells. We reveal that the role of HR in DNA DSB repair is hidden unless NHEJ is disabled. In addition, the types of chromosome aberrations observed after IR in SCID cells expressing the mutant hRAD54 reveal error-prone repair, showing a predominance of unresolved, incorrectly joined, chromatid exchange aberrations.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Transformed fibroblasts from ST. SCID, obtained in our laboratory (29) and the genetically matched parent, C.B-17, were grown in DMEM containing 10% FCS. S/MHB-2, S/mut-4, S/mut-6, C/MHB-2, C/mut-1, and C/mut-3 were grown in the same medium supplemented with 500 µg/ml Geneticin (Life Technologies, Inc.).

Subcloning.
The point mutant hHR54 cDNA was copied using PCR in such a way as to create KpnI (5') and XbaI (3') restriction sites in the cDNA sequence. The primers designed were hR54F-KpnI (5'-AAGGTACCATGAGG AGGAGCTTGGC TCCCAGCCAG-3') and hH54R-XbaI (5'-AATCTAGAGCGCGGAGG CCCCGCTGT TCCTCATGAGA-3'). The cDNA was subcloned into the pcDNA3.1-Myc/His B expression vector (Invitrogen), and expanded in Top 10F’ cells (Invitrogen). Maxipreps were performed using the QIAGEN protocol.

Mammalian Cell Transfections.
Prior to transfection, Myc-His B and hHR54-GRT/Myc-His B vectors were linearized with SSPI endonuclease. The digestion mix was heat inactivated, ethanol precipitated, and resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Three hundred µl of ST. SCID and C.B-17 fibroblasts at 1 x 107 cells/µl were mixed with 5 µl of linearized DNA in a 2-mm electroporation cuvette and electroporated at 960 µF and 250 V (Bio-Rad). Transfections were plated and incubated overnight at 37°C. The following day, medium containing 500 µg/ml Geneticin was added. The 2nd day after electroporation, cells were trypsinized and plated at various concentrations. Eleven to 12 days after transfection one clone/dish was chosen for clonal expansion.

Colony Survival Assays.
Serial dilutions of cell lines were made and plated in 100-mm2 dishes 3–4 h prior to treatment with IR or MMC. For MMC sensitivity experiments, cells were treated for 1 h. For radiation sensitivity experiments, cells were irradiated in a 137Cs-irradiator at a dose rate of ~1.49 Gy/min. All of the dishes were incubated for 9–11 days and stained with crystal violet for colony formation. For each dose and dilution, cells were plated in triplicate. Each experiment was performed a minimum of two times. Statistical analysis (mean with SE) and curve-fitting were performed using Prism Graphpad software.

SCE Analysis.
Bromodeoxyuridine (10-5 M) was added to foil-covered cell cultures for approximately two cell cycles (24 h). Four h prior to harvest, Colcemid (0.1 µg/ml) was added. Cells were harvested and dropped using standard conditions (0.075 M KCl for 30 min at 37°C, followed by three fixations in methanol:acetic acid 3:1, v/v). Slides were aged for 2 h in a 60°C oven prior to staining. For differential staining of sister chromatids, slides were immersed in Hoechst 33258 dye (0.25 mg/ml) for 25 min. Slides were then UV-irradiated under a layer of citric acid buffer on a slide warmer at 50°C for 11/2 h. Slides were rinsed, dried, and stained with Giemsa for 7 min.

DSB Repair Assay.
DSB repair analysis was performed as a variation of experiments described elsewhere (43) . Fibroblasts were seeded at 4 x 106 cells in T-175 flasks. One day later, the medium was changed to contain [14C]thymidine at a final concentration of 0.02 µCi/ml. Twenty-four h later, the medium was replaced with nonradioactive medium for ~24 h. Monolayers of cells were trypsinized and resuspended in PBS at 1 x 107 cells/ml in 1.5% low-melt agarose (at 40°C). The cell/agarose mixture was pipetted into 2-mm tubing and was allowed to cool on ice. Solidified cells in agarose were removed from the tubing, cut into 5-mm-long agarose plugs, and placed in cold PBS for irradiation. Plugs were irradiated in PBS at 40 Gy using a 137Cs-irradiator (dose rate of ~3.4 Gy/min) on ice. Asymmetric field inversion gel electrophoresis was performed as described previously (44) . Each time point was run three times per experiment. The amount of damage in the unirradiated control was subtracted from the different time points after IR in a given cell line, and each cell line was normalized to the IR-0 time point.

Cell Cycle Analysis.
Cell cycle analysis was performed as per manufacturer’s instructions using fluorescence-activated cell sorting (FACS)-Scan (Becton Dickinson). Twenty thousand cells were analyzed per sample.

Cell Culture and Radiation Exposure for Chromosome Aberration Analysis.
SCID cells transfected with empty vector (S/MHB-2) and two SCID cell lines transfected with mutant hRAD54 (S/mut-6, S/mut-3) were suspended at a concentration less than 1 x 106/ml and irradiated for 3.92 min (1.53 Gy/min.) for a dose equivalent to 6 Gy. After irradiation, cells were released into fresh medium and allowed to recover 24 h prior to harvest. Colcemid was added 4 h prior to harvest at a concentration of 0.1 µg/ml 24 h after irradiation cells were harvested and dropped using standard conditions (0.075 M KCl for 30 min at 37°C, followed by three fixations in methanol:acetic acid 3:1, v/v).

Chromosome Painting.
One µl of mouse digoxigenin- and FITC-labeled composite DNA probe specific for mouse chromosomes 2 and 8 (courtesy of Dr. James Tucker, Lawrence Livermore National Laboratory, Livermore, CA) was added to the hybridization mix (50% formamide, 2x SSC, 10% dextran sulfate, and 3.5 µl of mouse Cot-1), for a total volume of 15 µl. Hybridization and washes were performed as described by Spruill et al., (45) . Slides were counterstained with propidium iodide containing Vectorshield (Vector Laboratories, Burlingame, CA) before viewing.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To impair HR, a human RAD54 cDNA containing a mutation changing the lysine residue at position 189 to an arginine in the putative GKT Walker-type nucleotide-binding domain (hHR54-GRT) was transfected into wild-type C.B-17 and DNA-PK-deficient SCID cells. Previous work has shown that this mutant human RAD54 cDNA is unable to rescue the MMS-sensitive RAD54{Delta} phenotype in S. cerevisiae cells (10) , and overexpression of the equivalent mutant protein in S. cerevisiae leads to a dominant-negative phenotype in yeast (46) . In addition, RAD54 belongs to the SNF2/SWI2 family of proteins, and mutation of this ATPase site in these proteins disrupts their function (47) . The approximate location of the mutation in the human cDNA (Fig. 1BCitation , *) and the sites for the primers within the cDNA and pcDNA3.1/Myc-His B vector (Invitrogen) that produce a 1.4-kb fragment are shown in the diagram (Fig. 1B)Citation . To confirm expression of the mutant hRAD54 cDNA and verify stability of the transfectants from the vector, RT-PCR was performed throughout the course of experimentation. Fig. 1ACitation demonstrates amplification of the predicted 1.4-kb fragment (Lanes 3 and 5) in C.B-17 and SCID cell lines stably transfected with the mutant cDNA. In comparison, cDNA from cell lines transfected with vector only, C/MHB-2 and S/MHB-2, do not amplify the 1.4-kb fragment (Lanes 2 and 4). Primers to the proapoptotic cDNA, Bax, were used to confirm that the RT-PCR worked and the resulting PCR product was 163 bp.



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  		Fig. 1. A, confirmation of mutant hRAD54 expression. Top panel, total RNA from wild-type C.B-17 cells with empty vector (C/MHB-2, Lane 2), C.B-17 cells with mutant hRAD54 (C/mut-1, Lane 3), SCID cells with empty vector (S/MHB-2, Lane 4), and SCID cells with mutant hRAD54 (S/mut-6, Lane 5) were used for RT-PCR analysis, and the resulting amplified products were separated by agarose gel electrophoresis. A 1-kb ladder (Lane 1) was used to indicate the size of the resulting products. Bottom panel, amplification of Bax cDNA as a control for RT-PCR demonstrating equal expression in all of the cell lines. B, diagram showing hRAD54 cDNA with approximate location of mutation (*) that changes GKT domain into a GRT domain. Arrows, placement of forward and reverse primers within cDNA and vector used to produce the 1.4-kb product. {blacksquare}, regions of homology to known ATPases ("ATPase" Domains).

 
Sensitivity to MMC was determined in the cell lines to measure impairment of the HR pathway as interstrand cross-links appear to be repaired by HR (48) , and cell lines mutant for proteins in the RAD52 epistasis group in yeast demonstrate hypersensitivity to MMC (49) . An ~2-fold increase in sensitivity to MMC was observed in two separate cell lines of C.B-17 stably transfected with mutant hRAD54 (C/mut-1 and C/mut-3) compared with C.B-17 cells transfected with vector alone (C/MHB-2; Fig. 2ACitation ). Likewise, two SCID cell lines expressing mutant hRAD54 (S/mut-4 and S/mut-6) demonstrated an increased sensitivity to MMC when compared with SCID cells transfected with vector alone (S/MHB-2; Fig. 2BCitation ). As a control, RAD54-/- MEF cells (No. 2) were assayed for MMC sensitivity (Fig. 2C)Citation and showed a similar level of hypersensitivity to MMC when compared with RAD54-/- MEF cells transfected with wild-type RAD54 (No. 36). Although the various cell lines show differences in their baseline MMC sensitivity, in all cases when RAD54 function is affected, either by mutation or by deletion, an increase in MMC sensitivity is observed.



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  		Fig. 2. Dominant-negative action of mutant hRAD54 based on MMC sensitivity. A, MMC sensitivity for wild-type C.B-17 cell line transfected with empty vector (C/MHB-2) and two independently isolated wild-type lines transfected with mutant hRAD54 (C/mut-1 and C/mut-3) was determined using the colony survival assay. B, MMC sensitivity for a SCID cell line transfected with empty vector (S/MHB-2) and two independently isolated SCID lines transfected with mutant hRAD54 (S/mut-4 and S/mut-6) determined using the colony survival assay. C, MMC sensitivity in RAD54-/- MEFs (#2) and RAD54-/- MEFs expressing wild-type hRAD54 cDNA (#36) using the colony survival assay. Error bars, SE.

 
SCEs have been shown to be mediated by HR; thus, lower levels of SCEs have been observed in cells defective in RAD54 (50) . Therefore, to further confirm a defect in the HR pathway in our transfectants, SCE frequencies were determined for wild-type C.B-17 cells transfected with empty vector (C/MHB-2) and C.B-17 cells transfected with mutant hRAD54 (C-mut1). Wild-type C/MHB-2 cells showed roughly twice the level of SCEs compared with the cells expressing the mutant hRAD54 cDNA (24 ± 6 versus 11 ± 4, respectively), confirming the dominant-negative effect of the mutant hRAD54 cDNA on HR function. Thus, the increase in MMC sensitivity and reduced SCE frequencies in cell lines overexpressing the point mutant hRAD54 cDNA strongly indicate that the mutant protein induces a dominant-negative effect on the ability of these cells to perform HR.

Given that HR is impaired in the wild-type C.B-17 and SCID cell lines overexpressing mutant hRAD54, we wanted to determine whether this defect in HR contributes to the IR sensitivity of SCID cells. Wild-type C.B-17 transfectants expressing mutant hRAD54 (C/mut-1 and C/mut-3) did not demonstrate an increase in sensitivity to IR compared with wild-type cells transfected with vector alone (C/MHB-2; Fig. 3ACitation ). Likewise, RAD54-/- MEFs were not more sensitive to IR than are RAD54-/- MEFs stably transfected with wild-type hHR54 cDNA (Fig. 3B)Citation , although they exhibited hypersensitivity to MMC (Fig. 2C)Citation . The MEF cell lines show slightly higher levels of baseline radioresistance as compared with the transfected wild-type C.B-17 cells, but in both lines, a defect in RAD54 does not increase radiosensitivity as compared with each line’s respective control. In contrast, SCID transfectants overexpressing mutant hRad54 (S/mut-4 and S/mut-6) demonstrate an increase in IR sensitivity compared with SCID cells transfected with vector alone (S/MHB-2; Fig. 3ACitation ).



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  		Fig. 3. IR sensitivity of cell lines. A, survival of wild-type C.B-17 cells transfected with empty vector (C/MHB-2), wild-type C.B-17 cells transfected with mutant hRAD54 (C/mut-1, C/mut-3), SCID cells with empty vector (S/MHB-2), and SCID cells transfected with mutant hRAD54 (S/mut-3, S/mut-6) as measured by the colony survival assay. B, survival of RAD54-/- MEFs (#2) and RAD54-/- MEFs expressing wild-type hRAD54 cDNA (#36) as measured by the colony survival assay. Error bars, SE.

 
To further characterize the IR response, an assay for DNA DSB repair was performed on wild-type C.B-17 cells transfected with vector alone (C/MHB-2), C.B-17 cells transfected with mutant hRAD54 (C/mut-1), SCID cells transfected with empty vector (S/MHB-2), and SCID cells transfected with mutant hRAD54 (S/mut-6). Wild-type C/MHB-2 cells repaired ~85% of DNA DSBs by 6 h after 40 Gy of IR, whereas DNA-PK-deficient S/MHB-2 cells repaired only 57% of DSBs by 6 h postirradiation (Fig. 4)Citation . In comparison, SCID/RAD54-defective S/mut-6 cells were capable of repairing only 38% by 6 h after 40 Gy of IR. Wild-type cells transfected with mutant hRAD54 (C/mut-1) performed DSB repair similarly to wild-type cells transfected with vector alone (C/MHB-2, Fig. 4Citation ). Thus, defective DSB repair was exhibited with mutant hRAD54 expression only in a SCID background, not in a wild-type C.B-17 background, implying that a pathway involving RAD54 becomes more critical when NHEJ is impaired in mammalian cells.



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  		Fig. 4. DNA DSB repair assay. Asymmetric field inversion gel electrophoresis of cells IR in agarose plugs was used to determine the percentage of DNA DSB rejoined as a function of repair time for wild-type C.B-17 cells transfected with empty vector (C/MHB-2), C.B-17 transfected with mutant hRAD54 (C/mut-1), SCID cells transfected with empty vector (S/MHB-2), and SCID cells transfected with mutant hRAD54 (S/mut-6) after IR with 40 Gy. Values were determined from two independent experiments with three observations at each time point. Error bars, SE.

 
A surprising observation that warranted further investigation was the finding that wild-type cells transfected with empty vector (C/MHB-2) were more sensitive to MMC than were SCID cells transfected with empty vector (S/MHB-2; Fig. 2, A and BCitation ). To determine whether the MMC resistance was attributable to the mutant DNA-PKcs in SCID cells, we analyzed MMC sensitivity in SCID cells complemented with a human chromosome 8 fragment containing the DNA-PKcs gene (SCID/100E) and in SCID cells containing part of human chromosome 8 without the DNA-PKcs gene (SCID/50D; Ref. 31 ). Fig. 5Citation demonstrates that the DNA-PK-complemented SCID cell line, 100E, displays a MMC sensitivity similar to that of the wild-type C.B-17 cell line. In comparison, DNA-PK-deficient 50D cells show an increased resistance to MMC similar to that of SCID cells. These results suggest that the HR pathway of cross-link repair is more efficient in mammalian cells when the NHEJ pathway is defective. A similar resistance to MMS was observed by Takata et al. (15) in KU70-/- (NHEJ-defective) cells compared with wild-type DT40 cells.



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  		Fig. 5. MMC resistance in SCID cells attributable to lack of DNA-PK. DNA-PK complemented SCID cells (100E) exhibit a wild-type degree of MMC sensitivity. SCID cells, transfected with chromosome fragment not containing DNA-PK (50D), show increased resistance to MMC, similar to that of untransfected SCID cells. Error bars, mean ± SE.

 
To determine the effect of impaired HR and NHEJ on chromosome repair, we looked at chromosome aberrations in both the SCID cells with empty vector and the double-mutant cells transfected with mutant hRAD54. Fifty metaphase spreads were scored for both irradiated and unirradiated samples of SCID cells transfected with empty vector (S/MHB-2) and two SCID lines transfected with the mutant hRAD54 (S/mut-3 and 6 designated #3 and #6, respectively, in Table 1Citation ). All of the cell lines had approximately equal modal numbers of chromosomes per metaphase (S/MHB-2, 48 ± 5.4; S/mut-6, 49 ± 10.0; S/mut-3, 47 ± 10.2). Spontaneous aberration frequencies varied between cell lines ranging from 0.22 aberrations per cell in S/mut-3 to 2.22 aberrations per cell in S/mut-6. After irradiation, double-mutant cells (S/mut-3 and S/mut-6) showed an ~3-fold higher aberration frequency compared with that of the cell line with vector alone (S/MHB-2). Inductions observed within each line after irradiation differed because all of the cell lines had distinct baseline aberration frequencies. After IR, S/MHB-2 showed a <2-fold induction (1.8) in aberrations, whereas S/mut-6 showed nearly a 3-fold induction (2.9), and S/mut-3 showed a greater than a 33-fold induction (33.1). Especially striking were the large number of unresolved chromatid exchange aberrations observed in the double-mutant cells (Fig. 6)Citation . In the majority of double-mutant cells (58% of S/mut-3 cells and 86% of S/mut-6 cells) incomplete chromatid exchange aberrations were observed that joined ~2–26 chromosomes together. These types of aberrations were observed only rarely in wild-type C.B-17 cells transfected with mutant hRAD54 (C/mut -1; <1% of cells) or in SCID cells transfected with empty vector (S/MHB-2; 2% of cells), and when present in these cell lines, the exchanges always involved four or less chromosomes (data not shown).


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  		Table 1 Spontaneous and induced chromosome aberration frequenciesa in double mutant and control cells

 


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  		Fig. 6. Fluorescence in situ hybridization analysis of chromosome aberrations in SCID cells transfected with mutant hRAD54 after exposure to 6 Gy of IR. A, B, D, and E, incomplete chromatid exchange aberrations observed at x100. C, a typical metaphase spread at x60 with numerous incomplete chromatid exchange aberrations.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a dominant-negative system, we examined the roles of HR and NHEJ in mammalian DNA DSB repair. When mutant hRAD54 was expressed in wild-type C.B-17 cells, a hypersensitivity to MMC and a decrease in SCE frequency was observed; however, no change in IR sensitivity as compared with controls was noted. The increased MMC sensitivity and decreased SCE frequency indicate that we have negatively altered HR function in these cells. Although we cannot rule out that there is another function (other than HR) for RAD54, or that our dominant-negative hRAD54 has affected something other than HR (which happens to also affect DNA DSB repair), our dominant-negative hRAD54 appears to be specific to RAD54 function because our cells behave similar to the SCID/RAD54-/- whole animals described by Essers et al. (17) . In addition, the results that we observe in our dominant-negative system are also consistent with the findings in RAD54-/- MEFs, which fail to show increased sensitivity to IR but show increased MMC sensitivity compared with that of control RAD54-/- MEFs expressing wild-type hHR54 cDNA. Thus, we conclude that HR has been impaired; however, impairment of HR does not affect IR sensitivity. Therefore, in NHEJ functional, differentiated mammalian cells, HR plays no role, or plays a very minor role, in the repair of IR damage and in DSB repair. In all of the previously studied higher eukaryotic systems (ES cells, DT40 chicken cells, and Drosophila) a defect in RAD54 increased IR sensitivity compared with wild-type cells (14) . However, a report by Esssers et al., (17) reveals that RAD54-/- embryonic and adult differentiated cells respond differently to IR. Mouse embryonic RAD54-/- cells exhibit IR sensitivity, whereas adult RAD54-/- mice fail to show any sensitivity. Thus, DT40 chicken cells and Drosophila appear more similar to mouse embryonic cells in terms of the relative importance of HR in repair of IR damage, which is clearly different from what we observe in differentiated eukaryotic cells. This finding is perhaps expected given that both ES and DT40 cells exhibit higher levels of HR and different cell cycle characteristics (higher population in S-G2 phase and a lower population in G1 in asynchronously growing cultures) compared with our differentiated fibroblast cells.

A reciprocal pattern is observed in S. cerevisiae, which uses the HR pathway predominately (26 , 27 , 51 , 52) . A complete deletion mutant of HDF1, the S. cerevisiae homologue of the gene-encoding mammalian Ku70, did not show any sensitivity to IR. However, in a RAD52 mutant background in which HR is impaired, additional sensitization in response to IR was observed, leading to the conclusion that HDF1 plays a role in a non-HR repair process that can only be observed when HR is disabled. Takata et al. (15) created RAD54-/-, KU70-/-, and RAD54-/-/KU70-/- cells from the chicken B-cell line DT40. In these cell lines, the {gamma}-radiation sensitivity of the double-mutant cells was profoundly greater than that of either single mutant, indicating two complimentary repair pathways. These researchers also demonstrated that RAD54-/- chicken cells were more sensitive to IR than are KU70-/- chicken cells, indicating HR as the primary DSB repair pathway and NHEJ as a secondary pathway, similar to what was observed in yeast. In fact, KU70-/- chicken cells display an increased resistance to IR compared with wild-type chicken cells at higher doses. These researchers interpret these results as being attributable to stimulation of HR in cells defective in NHEJ. In contrast to what was observed in yeast and chicken cells, we find that the NHEJ pathway predominates over HR in the repair of IR-induced damage and in DSB repair in mammalian differentiated cells.

Although no increase in radiation sensitivity was observed in a wild-type, C.B-17 background, expression of dominant-negative hRAD54 in a SCID background resulted in an increase in radiation sensitivity. The implications of this are 2-fold. First, SCID cells, which are defective in DNA-PKcs, a protein involved in NHEJ, are more sensitive to IR than wild-type C.B-17 cells. Thus, to observe an augmentation of this radiosensitivity strongly implies that DNA-PKcs and RAD54 are part of two separate pathways of DNA DSB repair. These results are confirmed in the DSB break repair assay. Second, mutant hRAD54 only affects IR sensitivity in a SCID mutant background, not in a wild-type C.B-17 background, indicating that HR becomes more crucial when NHEJ is impaired in differentiated mammalian cells. Therefore, in mammalian fibroblasts, the importance of HR is uncovered only in NHEJ-deficient cells. In addition, an increase in resistance to MMC is observed in NHEJ-deficient cells (Fig. 2, A and B)Citation , with SCID cells containing empty vector (S/MHB-2) exhibiting more resistance to MMC than wild-type C.B-17 cells containing empty vector (C/MHB-2). Correspondingly, SCID cells complemented for DNA-PK (100E) show a sensitivity to MMC (Fig. 5)Citation similar to wild-type C.B-17 cells, whereas DNA-PK-deficient cells (50D) show an increased resistance to MMC similar to that of SCID.

Higher chromosome aberration frequency inductions were observed in the double-mutant cells after IR when compared with the SCID cells containing the vector alone. Particularly striking was the large number of unresolved complex chromatid exchange-type aberrations observed in the double-mutant cells (Fig. 6)Citation . These incomplete exchange aberrations, resulted in multiple chromatid attachments among several chromosomes (up to 26 chromosomes in one case). Chromatid aberrations are often indicative of damage occurring in the preceding S-G2 phase of the cell cycle. Interestingly, no comment was made of these types of incomplete chromatid aberrations in the chromosome analysis of chicken DT40 cells deficient in Ku70 and RAD54, although the authors (15) did note an overall increase in chromatid aberrations. The lack of this finding in chicken DT40 cells could be attributable to the differing level of importance of the HR pathway in the two cell types (as was previously mentioned) or because Ku was deficient in these cells rather than DNA-PK as in our cells. The error-prone repair observed in our double-mutant cells could represent incomplete repair attributable to the presence of an aberrant hRAD54 protein, or it could be the result of a low-fidelity repair process (perhaps an additional pathway initiated in the absence of both the NHEJ and HR). In both yeast and T4 bacteriophage, other repair pathways besides the two predominant pathways have been observed (53 , 54) , but the exact participants in these alternative pathways have yet to be elucidated.

Numerous unresolved complex chromatid exchange aberrations have also been observed in ataxia telangiectasia cells exposed to camptothecin, a topoisomerase I poison specific to S phase cells (55) . These researchers concluded that in the absence of a functional ATM protein DSBs formed were processed abnormally, resulting in many heterologous chromatid exchanges that were observed only infrequently in camptothecin-treated wild-type cells. The fact that we also observed high levels of incorrectly repaired S-phase damage does not imply that our double-mutant cells have a defect only in S-phase repair. Observing primarily error-prone S-phase damage could reflect the fact that we harvested cells 24 h after IR and, because of the slow recovery of these cells, the majority of the cells that made it to metaphase by harvest were ones in S-G2 phase at the time of IR. Further analysis harvesting cells at longer time periods after IR would be necessary to determine whether error-prone repair is also observed in other phases of the cell cycle in these double-mutant cells.

Unresolved chromatid exchanges are also frequently observed in FA patients’ cells when treated with a DNA cross-linker, diepoxybutane (56) . FA is an inherited disorder displaying chromosome instability and extreme cancer proneness (57) . Cells from FA patients show extreme sensitivity to cross-linking agents, and the underlying defect has been hypothesized to be in a DNA-repair pathway. Although no obvious DNA-repair defect has been firmly established in FA, recent reports indicate possible defects in NHEJ and in the fidelity of repair (58, 59, 60) . The similarity between aberrations in FA cells and those we describe for our double-mutant cells may suggest that an in-depth investigation of the NHEJ and HR pathways in FA cells might prove illuminating. Perhaps various FA complementation group proteins provide an interface for the NHEJ and HR pathways and aid in coordinating DNA repair after DNA damage.

In summary, by constructing a SCID/RAD54 double-mutant cell line, we have created a unique genetic system to study the involvement of two pathways, NHEJ and HR, in DNA DSB repair in response to IR. Our finding that HR plays a minor role in DNA DSB repair unless NHEJ is defective confirms the notion that NHEJ is the primary DNA DSB repair pathway in mammalian fibroblast cells. This result is in contrast to previously published reports in mouse ES cells, chicken DT40 cells, and Drosophila, which all show a role for HR even in NHEJ functional cells. In addition, our studies reveal HR to be more efficient in repairing MMC damage in the absence of NHEJ. And lastly, data from chromosome aberration studies after IR reveal that a defect in both HR and NHEJ, results in severe genetic instability and error-prone repair.


   ACKNOWLEDGMENTS
 
We thank Roland Kanaar and Jeroen Essers for RAD54-/- MEFs (no. 2), RAD54-/- MEFs expressing wild-type hRAD54 cDNA (no. 36), and hHR54 cDNA, as well as for helpful discussions. We also thank James Tucker for the mouse 2:8 probe for fluorescence in situ hybridization analysis and Jim Evans, Vikky Yamazaki, and Susannah Green for their assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by PHS Grant 5R29 CA75404 awarded by the National Cancer Institute (to J. M. P. and L. M. F.), by the Katherine McCormick fund (to J. M. P.), NIH Grant CA80207 (to J. M. P.), and by the Director, Office of Science, Office of Basic Energy Sciences, of the United States Department of Energy under Contract No. DE-AC03-76SF00098 (to J. M. P.). Back

2 J. M. P. and L. M. F. contributed equally to this work. Back

3 Present address: Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 74-157, Berkeley, CA 94720. Back

4 To whom requests for reprints should be addressed, at CCSR, South, Room 1245C, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5152. Email: cordula{at}stanford.edu Back

5 The abbreviations used are: DSB, double-strand break; NHEJ, nonhomologous end joining; SCID, severe combined immunodeficient; HR, homologous recombination; SCE, sister chromatid exchange; MMC, mitomycin-C; IR, ionizing radiation; ES, embryonic stem; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; MMS, methanesulfonate; RT-PCR, reverse transcription-PCR; FA, Fanconi anemia. Back

Received 9/ 5/00. Accepted 1/17/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baumann P., West S. C. Role of human RAD51 protein in homologous recombination and double-stranded-break repair. Trends Biochem. Sci., 23: 247-251, 1999.
  2. Haber J. E. DNA recombination: the replication connection. Trends Biochem. Sci., 24: 271-275, 1999.[Medline]
  3. Petes T. D. Malone R. E. Symington L. S. eds. . The Molecular and Cellular Biology of the Yeast Saccharomyces, 1: 407-522, Cold Spring Harbor Laboratory Cold Spring Harbor, NY 1991.
  4. Petrini J. H. J., Bressan D. A., Yao M. S. The RAD52 epistasis group in mammalian double strand break repair. Semin. Immunol., 9: 181-188, 1997.[Medline]
  5. Shinohara A., Ogawa H., Matsuda Y., Ushio N., Ikeo K., Ogawa T. Cloning of human, mouse, and fission yeast recombination genes homologous to RAD51 and recA. Nat. Genet., 4: 239-243, 1993.[Medline]
  6. Thacker J. The role of homologous recombination processes in the repair of severe forms of DNA damage in mammalian cells. Biochimie (Paris), 81: 77-85, 1999.[Medline]
  7. Morita T., Yoshimura Y., Yamamoto A., Murata K., Mori M., Yamamoto H., Matsushiro A. A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 genes. Proc. Natl. Acad. Sci., 90: 6577-6580, 1993.[Abstract/Free Full Text]
  8. Muris D. F. R., Bezzubova O., Buerstedde J-M., Vreeken K., Bajajee A. S., Osgood C. J., Troelstra C., Hoeijmakers J. H. J., Ostermann K., Schmidt H., Natarajan A. T., Eeken J. C. J., Lohman P. H. M., Pastink A. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination. Mutat. Res., 315: 295-305, 1994.[Medline]
  9. Shen Z., Denison K., Lobb R., Gatewood J. M., Chen D. J. The human and mouse homologs of the yeast RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse testis. Genomics, 25: 199-206, 1995.[Medline]
  10. Kanaar R., Troelstra C., Swagemakers S. M. A., Essers J., Smit B., Franssen J-H., Pastnik A., Bezzubova O. Y., Buerstedde J-M., Clever B., Heyer W-D., Hoeijmakers J. H. J. Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation. Curr. Biol., 6: 828-838, 1996.[Medline]
  11. Lim D. S., Hasty P. A mutation in mouse Rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell. Biol., 16: 7133-7143, 1996.[Abstract]
  12. Tsuzuki T., Fujii Y., Sakumi K., Tominaga Y., Nakao K., Sekiguchi M., Matsushiro A., Yoshimura Y., Morita T. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA, 93: 6236-6240, 1996.[Abstract/Free Full Text]
  13. Rijkers T., VanDen Ouweland J., Morolli B., Rolink A. G., Baarends W. M., Van Sloun P. P., Lohman P. H., Pastink A. Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol. Cell. Biol., 18: 6423-6429, 1998.[Abstract/Free Full Text]
  14. Essers J., Hendriks R. W., Swagemakers S. M. A., Troelstra C., de Wit J., Bootsma D., Hoeijmakers J. H. J., Kanaar R. Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination. Cell, 89: 195-204, 1997.[Medline]
  15. Takata M., Sasaki M. S., Sonoda E., Morrison C., Hashimoto M., Utsumi H., Yamaguchi-Iwai Y., Shinohara A., Takeda S. Homologous recombination and nonhomologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J., 17: 5497-5508, 1998.[Medline]
  16. Kooistra R., Pastink A., Zonneveld J. B., Lohman P. H., Eeken J. C. The Drosophila melanogaster Dm RAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage. Mol. Cell. Biol., 19: 6269-6275, 1999.[Abstract/Free Full Text]
  17. Essers J., van Steeg H., de Wit J., Swagemakers S. M. A., Vermeij M., Hoeijmakers J. H. J., Kanaar R. Homologous and nonhomologous recombination differentially affect DNA damage repair in mice. EMBO J., 19: 1703-1710, 2000.[Medline]
  18. Roth D., Wilson J. Illegitimate recombination in mammalian cells Kucherlapati R. Smith G. R. eds. . Genetic Recombination, : 621-653, American Society for Microbiology Washington DC 1988.
  19. Tsukamoto Y., Ikeda H. Double-strand break repair mediated by DNA end-joining. Genes Cells, 3: 135-144, 1998.[Abstract]
  20. Li Z., Otevrel T., Gao Y., Cheng H-L., Seed B., Stamato T. D., Taccioli G. E., Alt F. W. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell, 83: 1079-1089, 1995.[Medline]
  21. Jeggo P. DNA-PK: at the crossroads of biochemistry and genetics. Mutat. Res., 384: 1-14, 1997.[Medline]
  22. Wilson T. E., Grawunder U., Lieber M. R. Yeast DNA ligase IV mediates nonhomologous DNA end joining. Nature (Lond.), 388: 495-498, 1997.[Medline]
  23. Critchlow S. E., Bowater R. P., Jackson S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr. Biol., 7: 588-598, 1997.[Medline]
  24. Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature (Lond.), 388: 492-498, 1997.[Medline]
  25. Weaver D. T. What to do at an end: DNA double-stand-break repair. Trends Genet., 11: 388-392, 1995.[Medline]
  26. Siede W., Friedl A. A., Dianova I., Eckardt-Schupp F., Friedberg E. C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination. Genetics, 142: 91-102, 1996.[Abstract]
  27. Boulton S. J., Jackson S. P. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J., 15: 5093-5103, 1996.[Medline]
  28. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A., Jackson S. P. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell, 80: 813-823, 1995.[Medline]
  29. Biedermann K. A., Sun J., Giaccia A. J., Tosto L. M., Brown J. M. Scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc. Natl. Acad. Sci., 88: 1394-1397, 1991.[Abstract/Free Full Text]
  30. Peterson S. R., Kurimasa A., Oshimura M., Dynan W. S., Bradbury E. M., Chen D. J. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells. Proc. Natl. Acad. Sci. USA, 92: 3171-3174, 1995.[Abstract/Free Full Text]
  31. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent protein kinase (p350) as a candidate gene for the murine SCID defect. Science (Washington DC), 267: 1178-1183, 1995.[Abstract/Free Full Text]
  32. Fulop G. M., Phillips R. A. The scid mutation in mice causes a general defect in DNA repair. Nature (Lond.), 347: 479-482, 1990.[Medline]
  33. Hendrickson E. A., Qin X. Q., Bump E. A., Schatz D. G., Oettinger M., Weaver D. T. A link between double-strand break-related repair and V(D)J recombination: the scid mutation. Proc. Natl. Acad. Sci. USA, 88: 4061-4065, 1991.[Abstract/Free Full Text]
  34. Chang C., Biedermann K. A., Mezzina M., Brown J. M. Characterization of the DNA double strand break repair defect in scid mice. Cancer Res., 53: 1244-1248, 1993.[Abstract/Free Full Text]
  35. Kirchgessner C. U., Tosto L. M., Biedermann K. A., Kovacs M., Araujo D., Stanbridge E. J., Brown J. M. Complementation of the radiosensitive phenotype in severe combined immunodeficient mice by human chromosome 8. Cancer Res., 53: 6011-6016, 1993.[Abstract/Free Full Text]
  36. Danska J. S., Holland D. P., Mariathasan S., Williams K. M., Guidos C. J. Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol. Cell. Biol., 16: 5507-5517, 1996.[Abstract]
  37. Blunt T., Gell D., Fox M., Taccioli G. E., Jackson S. P., Lehmann A. R., Jeggo P. A. Identification of a nonsense mutation in the carboxy-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. USA, 93: 10285-10290, 1996.[Abstract/Free Full Text]
  38. Araki R., Fujimori A., Hamatani K., Mita K., Saito T., Mori M., Fukumura R., Morimyo M., Muto M., Itoh M., Tatsumi K., Abe M. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc. Natl. Acad. Sci. USA, 94: 2438-2443, 1997.[Abstract/Free Full Text]
  39. Gao Y., Chaudhuri J., Zhu C., Davidson L., Weaver D. T., Alt F. W. A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for Ku in V(D)J recombination. Immunity, 9: 367-376, 1998.[Medline]
  40. Jhappan C., Morse H. C., Fleischmann R. D., Gottesman M. M., Merlino G. DNA-Pkcs: a T-cell tumor suppressor encoded at the mouse scid locus. Nat. Genet., 17: 483-486, 1997.[Medline]
  41. Taccioli G., Amatucci A. G., Beamish H. J., Gell D., Xiang X. H., Arzayus M. I., Priestley A., Jackson S. P., Rothstein A. M., Jeggo P. A., Herrera V. L. M. Targeted disruption of the catalytic subunit of the DNA-Pk gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity, 9: 355-366, 1998.[Medline]
  42. Liang F., Han M., Romanienko P., Jasin M. Homology-directed repair is a major double strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. USA, 95: 5172-5177, 1998.[Abstract/Free Full Text]
  43. Stamato T. D., Denko N. Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells. Radiat. Res., 121: 196-205, 1990.[Medline]
  44. Rydberg B., Lobrich M., Cooper P. K. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. I. Pulsed-field gel electrophoresis method. Radiat. Res., 139: 133-141, 1994.[Medline]
  45. Spruill M. D., Ramsey M. J., Swiger R. R., Nath J., Tucker J. D. The persistence of aberrations in mice induced by {gamma} radiation as measured by chromosome painting. Mutat. Res., 356: 135-145, 1995.
  46. Clever B., Schmuckli-Maurer J., Sigrist M., Glassner B. J., Heyer W. Specific negative effects resulting from elevated levels of the recombinational repair protein Rad54p in Saccharomyces cerevisiae. Yeast, 15: 721-740, 1999.[Medline]
  47. Cote J., Quinn J., Workman J. L., Peterson C. L. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science (Washington DC), 265: 53-60, 1994.[Abstract/Free Full Text]
  48. Thompson L. H. Evidence that mammalian cells possess homologous recombinational repair pathways. Mutat. Res., 363: 77-88, 1996.[Medline]
  49. Abe F. W., Wada M., Kohno K., Kuwano M. Altered drug sensitivities to anticancer agents in radiation sensitive DNA repair deficient yeast mutants. Anticancer Res., 14: 1807-1810, 1994.[Medline]
  50. Sonoda E., Sasaki M. S., Morrison C., Yamaguchi-Iwai Y., Takata M., Takeda S. Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol. Cell. Biol., 19: 5166-5169, 1999.[Abstract/Free Full Text]
  51. Milne G. T., Jin S., Shannon K. B., Weaver D. T. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol., 16: 4189-4198, 1996.[Abstract]
  52. Tsukamoto Y., Kato J. I., Ikeda H. Hdf1, a yeast Ku-protein homologue, is involved in illegitimate recombination, but not in homologous recombination. Nucleic Acids Res., 24: 2067-2072, 1996.[Abstract/Free Full Text]
  53. Geourge J. W., Kreuzer K. N. Repair of double-strand breaks in bacteriophage T4 by a mechanism that involves extensive DNA replication. Genetics, 143: 1507-1520, 1996.[Abstract]
  54. Malkova A., Ivanov E. L., Haver J. E. Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc. Natl. Acad. Sci. USA, 93: 7131-7136, 1996.[Abstract/Free Full Text]
  55. Johnson R. T., Gotoh E., Mullinger A. M., Ryan A. J., Shiloh Y., Ziv Y., Squires S. Targeting double-strand breaks to replicating DNA identifies a subpathway of DSB repair that is defective in ataxia-telangiectasia cells. Biochem. Biophys. Res. Commun., 261: 317-325, 1999.[Medline]
  56. Auerbach A. D. Fanconi anemia and leukemia: tracking the genes. Leukemia, 6: 1-4, 1992.
  57. Wright E. G. Inherited and inducible chromosomal instability: a fragile bridge between genome integrity mechanisms and tumorigenesis. J. Pathol., 187: 19-27, 1999.[Medline]
  58. Escarceller M., Buchwald M., Singleton B. K., Jeggo P., Jackson S. P., Moustacchi E., Papadopoulo D. Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining. J. Mol. Biol., 279: 375-385, 1998.[Medline]
  59. Escareller M., Rousset S., Moustacchi E., Papadopoulo D. The fidelity of double strand breaks processing is impaired in complementation groups B and D of Fanconi anemia, a genetic instability syndrome. Somatic Cell Mol. Genet., 23: 401-411, 1997.[Medline]
  60. Smith J., Andrau J. C., Kallenbach S., Laquerbe A., Doyen N., Papadopoulo D. Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia. J. Mol. Biol., 281: 815-825, 1998.[Medline]



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