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Enhanced fidelity for rejoining radiation-induced DNA double-strand br
http://www.100md.com 《核酸研究医学期刊》
     Fachrichtung Biophysik, Universit?t des Saarlandes, D-66421 Homburg/Saar, Germany

    *To whom correspondence should be addressed. Tel: +49 6841 1626202; Fax: +49 6841 1626160; Email: markus.loebrich@uniklinik-saarland.de

    Present address:

    Kai Rothkamm, Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK

    ABSTRACT

    The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G2 than in G1, suggesting that DSB rejoining in G2 proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G1 and G2 compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G2 occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G1 and G2. We propose that structural characteristics of G2 phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.

    INTRODUCTION

    Of the many types of damage that arise in DNA, the most critical lesions are DNA double-strand breaks (DSBs). They are produced by ionizing radiation (IR) and certain chemicals and arise endogenously during DNA replication and during developmental processes such as V(D)J recombination and meiotic crossing-over. If left unrepaired, DSBs can induce permanent cell cycle arrest, apoptosis or mitotic cell death caused by loss of genomic material (1). If repaired incorrectly, they can trigger carcinogenesis through translocations, inversions or deletions of genomic material (2,3).

    Higher eukaryotic cells primarily utilize two genetically separable pathways for DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ repairs DSBs without a requirement for extensive sequence homology at the break ends. It involves the XRCC4–DNA ligase IV complex and the DNA-dependent protein kinase (DNA-PK) holoenzyme, which consists of the DNA end-binding heterodimer Ku70/Ku80 and the catalytic subunit DNA-PKcs (4–6). Defects in any of these proteins confer IR sensitivity and defective DSB rejoining (7–10). Functional NHEJ has been described as a caretaker of chromosomal integrity (11,12) and increased levels of radiation-induced chromosomal aberrations have been observed in cells deficient in NHEJ (13–16). However, NHEJ has the potential to generate genomic rearrangements by joining incorrect break ends (17,18). The probability for this type of mis-rejoining has been proposed to be influenced by the structural characteristics of the chromatin, which may limit the diffusibility of break ends (19).

    HR, which utilizes an undamaged homolog to faithfully restore the sequence at the break site (20), appears to be active predominantly in the S and G2 phases of the cell cycle, when a sister chromatid is available (21). HR is predicted to rejoin breaks accurately since heteroallelic recombination has been reported to be a very rare event (22) and since the outcome of HR in mammalian cells is predominantly gene conversion without crossing-over (23). Thus, HR should not contribute to genomic rearrangements. We have recently assessed the relative contribution of NHEJ and HR to DSB repair in different cell cycle phases by enumerating -H2AX foci after low (1 Gy) X-ray doses (24). We found that NHEJ is important for cell survival and DSB repair throughout the cell cycle whereas HR preferentially contributes to DSB repair and survival in late S/G2 (24).

    We have previously reported that the induction and correct rejoining of DSBs can be analyzed by evaluating large genomic restriction fragments in irradiated and repair-incubated mammalian cells (18,19,25). Our assay to monitor correct DSB rejoining involves restriction enzyme digestion of genomic DNA after irradiation and repair incubation, followed by separation of the restriction fragments using pulsed-field gel electrophoresis (PFGE) and subsequent detection of a specific fragment by Southern hybridization. DSB induction is assessed by a decrease in intensity of the hybridizing restriction fragment band and correct DSB rejoining is monitored by restriction fragment reconstitution. Since rejoining of correct DSB ends (i.e. ends that were linked before irradiation) reconstitutes the original restriction fragment whereas joining of incorrect break ends generates aberrant restriction fragments that are smaller or larger than the original fragment, the rejoining of correct DSB ends, also termed correct DSB rejoining, can be assessed by measuring the extent of reconstitution of the original restriction fragment band. However, in this assay correct rejoining might also include events involving gain or loss of short DNA sequences. Correct DSB rejoining is taken to represent a high fidelity process and is contrasted with the joining of incorrect break ends, also termed mis-rejoining, that includes, for example, translocations or inversions. The aim of this work was to evaluate the fidelity of DSB rejoining in different phases of the mammalian cell cycle.

    MATERIALS AND METHODS

    Cell synchronization, irradiation and lysis

    Chinese hamster ovary (CHO) cells (K1, AA8, xrs-6, V3 and irs1SF) were grown in MEM supplemented with 10% fetal bovine serum and antibiotics. Cells were seeded at a density of 8 x 104 cells/cm2 and grown for 3–4 days to obtain G1 phase cells; 4 x 104 G1 phase cells/cm2 were grown for 16 h in medium containing 1 μg/ml aphidicolin. Under these conditions, cells accumulate at the G1/S border. The aphidicolin-containing medium was then removed and the cells were incubated for 6 h (6.5 h for irs1SF cells) in fresh medium to obtain G2 phase cells. Measurements of cell cycle distributions were performed with a FACScan flow cytometer and CellQuest software (BD Biosciences, San Diego, CA). Cells were harvested, resuspended in phosphate-buffered saline (PBS), fixed with 70% ethanol at –20°C and stained with propidium iodide/RNase A. X-irradiation was performed at 95 kV and 25 mA with a 1.3 mm aluminum filter and a dose rate of 6 Gy/min as determined by chemical dosimetry. Cells were irradiated in flasks filled with ice-cold PBS, which was replaced by pre-warmed medium for repair incubation. Control samples were sham-irradiated in all experiments. Cell lysis and DNA extraction were performed as previously described (26). Briefly, cells were harvested, embedded in agarose plugs (6 x 105 G1 phase cells/plug and 3 x 105 G2 phase cells/plug) and lysed with pronase E at 50°C for 2 days.

    PFGE analysis

    To determine total DSB rejoining (FAR assay), DNA was separated by PFGE with a CHEF DR system (Bio-Rad, Hercules, CA) in 0.8% agarose gels. The gels were run at 14°C with linearly increasing pulse times from 50 to 5000 s over 65 h at a field strength of 1.5 V/cm. Gels were stained with ethidium bromide, images were obtained with a charge-coupled device camera under UV illumination and the fraction of DNA below the well was quantified with the AIDA software (Raytest, Straubenhardt, Germany). Experiments measuring the fraction of DNA below the well as a function of dose were performed in parallel to repair experiments, with the results serving as a calibration to obtain relative numbers of remaining DSBs from the fraction of DNA below the well in the repair samples. To determine restriction fragment reconstitution (i.e. the level of correct DSB rejoining), DNA was digested with the rare-cutting restriction enzyme MluI prior to electrophoresis. Agarose gels (0.8%) were run at 16°C for 46 h at 3 V/cm with pulse times increasing linearly from 40 to 800 s. After PFGE separation, the DNA was partly depurinated and transferred to a nylon membrane (Hybond XL; Amersham Pharmacia) by vacuum alkaline transfer. Pre-hybridization and hybridization were performed in 20 ml of 5x SSPE, 5x Denhardt’s, 3% SDS and 100 μg/ml sonicated denaturated salmon sperm DNA at 65°C in hybridization bottles. Chinese hamster dihydrofolate reductase cDNA (ATCC, catalog no. 77273), which detects a 1.9 Mb MluI restriction fragment of hamster DNA (26,27), was labeled by random priming with dCTP. Membranes were wrapped in Saran wrap after washing and exposed for several days to an imaging screen. The screen was scanned by a phosphoimaging system and quantitative analysis was carried out using the AIDA software (Raytest). A detailed description of the assays and the evaluation procedures are published in Rothkamm and L?brich (26).

    RESULTS

    Similar yield of X-ray-induced DSBs in G1 and G2

    Parental AA8 and K1 cells, XRCC3-defective irs1SF (28), DNA-PKcs-defective V3 and Ku80-defective xrs-6 cells (4) were grown to confluence to obtain at least 90% G1 phase cells (Fig. 1, first row). To investigate DSB induction and rejoining in G2, confluent cells were subcultured and treated with aphidicolin for 16 h. Six hours (6.5 h for irs1SF cells) after release from the aphidicolin block, >80% of the cells were in late S/G2 (Fig. 1, fourth row). DSB induction was measured with the specialized PFGE assay described above by quantifying the intensity of the hybridization signal representing the intact restriction fragment (IRF), which is normalized to the total signal intensity of each lane (IT) to account for differences in DNA loading. After radiation exposure, IRF decreases in a dose-dependent manner (see Fig. 4, lanes 1–5). In this approach, the number of DSBs induced per restriction fragment equals the negative logarithm of the ratio of IRF/IT of the irradiated sample to IRF/IT of the control sample (26). In all cell lines, the yield of DSB induction (expressed as DSBs/Mb/Gy) was very similar for G1 and G2 phase cells (Fig. 2), indicating that DNA is equally susceptible to radiation-induced breakage in both cell cycle phases.

    Figure 1. DNA histograms of unirradiated and 80 Gy irradiated CHO cells synchronized in G1 (upper three rows) and G2 (lower three rows).

    Figure 4. Restriction fragment reconstitution in G1 and G2 phase cells as determined by Southern hybridization analysis. (Left) Southern hybridization images of MluI-digested DNA from G1 and G2 phase cells irradiated with 0, 20, 40, 60 and 80 Gy without repair or incubated for up to 24 h for repair after 80 Gy irradiation. (Right) Time course for the percentage of initial DSBs not correctly rejoined after repair. Error bars represent the SEM from two or three experiments. The data from the top two panels are redrawn in the bottom two panels in grey symbols for a direct assessment of the impact of NHEJ deficiency.

    Figure 2. Yields of DSBs as a function of radiation dose for G1 and G2 phase CHO cells as determined by Southern hybridization analysis of a 1.9 Mb restriction fragment (RF). Average induction rates are as follows (in 10–3 DSBs/Mb/Gy): AA8, G1 7.2, G2 7.8; K1, G1 6.4, G2 6.4; irs1SF, G1 7.4, G2 6.7; V3, G1 6.7, G2 6.6; xrs-6, G1 6.7, G2 6.3. Error bars represent the SEM from two or three experiments.

    Improved DSB rejoining in G2 phase NHEJ mutants

    Conventional PFGE analysis was applied to analyze the time course for total DSB rejoining after 80 Gy X-rays. AA8, K1 and the HR mutant line irs1SF repair DSBs with similar kinetics in G1 and G2 (Fig. 3). After a repair period of 24 h, nearly all DSBs are rejoined in all cell lines, indicating that DSB rejoining is very efficient in the absence of HR in both G1 and G2. The NHEJ mutants xrs-6 and V3, in contrast, exhibit a pronounced repair defect with 40% unrejoined DSBs in G1 (Fig. 3), consistent with previous measurements (18). Interestingly, only 10–20% unrejoined DSBs are observed in xrs-6 and V3 cells in G2, indicating that specific factors or conditions enhance the capacity for DSB rejoining in this cell cycle phase. This result is consistent with data from -H2AX analysis, which also indicate substantially improved DSB rejoining in G2 phase NHEJ mutants (24). Compared with wild-type cells and HR mutants, however, NHEJ-deficient cells exhibit impaired kinetics in G2 (Fig. 3).

    Figure 3. DSB repair in G1 and G2 phase cells as determined by conventional PFGE. (Left) Ethidium bromide images of DNA from G1 and G2 phase cells irradiated with 0, 20, 40, 60 and 80 Gy without repair or incubated for up to 24 h for repair after 80 Gy irradiation. Sc, Saccharomyces cerevisiae size standard. (Right) Time course for the percentage of initial DSBs remaining after repair. Error bars represent the SEM from two or three experiments. The data from the top two panels are redrawn in the bottom two panels in grey symbols for a direct assessment of the impact of NHEJ deficiency.

    Restriction fragment reconstitution is more efficient in G2 than in G1

    To investigate the fidelity of DSB rejoining in G1 and G2, the specialized PFGE approach was employed. For a quantitative analysis, the band intensity, IRF, of a repair sample is transformed into the number of DSBs (by taking the negative logarithm of the ratio of IRF/IT of the repair sample to IRF/IT of the control sample; see above) and this number is divided by the number of DSBs obtained in a sample irradiated with the same dose but not incubated for repair. This provides an estimate of the fraction of DSBs that have not been rejoined correctly (i.e. DSBs that have either remained unrejoined or have been mis-rejoined to generate an aberrant restriction fragment). Analysis of the time course for correct DSB rejoining after 80 Gy shows that repair-proficient AA8 and K1 cells in G1 rejoin 50–60% of the induced DSBs correctly (Fig. 4), consistent with previous observations in wild-type CHO cells (18,26,27). For the same cells synchronized in G2, 80–90% correct rejoining is observed. This indicates that the fidelity of DSB rejoining is significantly higher in G2 than in G1.

    We next investigated the fidelity of DSB rejoining in irs1SF, V3 and xrs-6 cells (Fig. 4). Importantly, in G1 as well as in G2, the kinetics of correct DSB rejoining for irs1SF cells are very similar to those found with wild-type cells, indicating that HR does not contribute significantly to correct DSB rejoining and is not responsible for the enhanced fidelity of DSB rejoining in G2 phase wild-type cells. Examination of the time course for restriction fragment reconstitution in NHEJ-deficient cells shows significantly improved band reconstitution in G2 compared with G1, similar to the result for total DSB rejoining obtained by conventional PFGE analysis. Moreover, in both cell cycle phases restriction fragment reconstitution is less efficient in the NHEJ mutants than in wild-type cells, indicating that NHEJ contributes to correct DSB rejoining in both G1 and G2.

    DISCUSSION

    We employed two different PFGE techniques to investigate DSB repair in different phases of the mammalian cell cycle. Conventional PFGE was employed to measure total DSB rejoining that encompasses both correct and incorrect rejoining events while a specialized PFGE approach was used to assess the induction and correct rejoining of DSBs. Using a similar strategy, we previously showed that NHEJ can effect incorrect DSB rejoining following high acute radiation doses (18) and that the extent of incorrect rejoining is lower in heterochromatic centromere regions than in average genomic DNA (19). Here, we analyze DSB rejoining in the G1 and G2 phases of wild-type CHO cells after 80 Gy X-irradiation. Consistent with published data (29,30), we observed a similar time course for total DSB rejoining in both phases of the cell cycle (Fig. 3). In contrast, correct DSB rejoining is substantially higher in G2 compared with G1 (Fig. 4). This finding indicates that G2-specific conditions, although not contributing to the time course of total DSB rejoining, enhance the fidelity of the rejoining process. We tested the possibility that HR contributes to this effect and did not observe any significant DSB repair defect in HR-deficient irs1SF cells in late S/G2 using either the conventional or the specialized PFGE technique. This finding is consistent with previous reports that HR-defective cells fail to exhibit a DSB repair defect in assays that require high radiation doses (31–34) but contrasts with results from -H2AX analysis after a dose of 1 Gy that uncovered a role of HR in DSB repair in G2 (24). It is possible that PFGE fails to uncover this contribution because of the necessity of using high doses and/or the limited sensitivity of this technique. However, even after a dose of 1 Gy, only a few DSBs remain unrepaired in HR-deficient cells. Hence, both techniques (PFGE at high doses and -H2AX analysis at low doses) demonstrate that the majority of the rejoining events in G2 involve HR-independent processes (24).

    Our data demonstrate that HR-independent processes rejoin DSBs with higher fidelity in G2 than in G1. The observation that NHEJ mutants exhibit impaired restriction fragment reconstitution in G1 and G2 demonstrates the importance of this pathway for correct DSB rejoining in both cell cycle phases. Additionally, DSB rejoining in the absence of DNA-PK proceeds with faster kinetics and higher fidelity in G2 than in G1. This finding is consistent with earlier studies with synchronized populations of NHEJ-deficient cells that show increased DSB rejoining in NHEJ mutants in G2 relative to G1 (35,36). Substantial DSB rejoining is also observed after 20 and 80 Gy in G2 phase populations of DT40 chicken cells deficient for both Ku70 and Rad54 (34; K. Rothkamm and M. L?brich, unpublished data), consistent with the idea that DSBs can be rejoined in the absence of these factors (37,38).

    A possible explanation for the increased fidelity of DSB rejoining in G2 involves structural differences between G1 and G2 phase chromatin. Such differences could be effected by the cohesin complex, which consists of the heterodimer SMC1 and SMC3 and at least two non-SMC subunits (Scc1/Mcd1/RAD21 and Scc3/SAs). In Saccharomyces cerevisiae, cohesin associates with specific regions near centromeres and along chromosome arms with a preference for AT-rich sequences. In the arms, cohesin distributes with a periodicity of 9–15 kb (39). Throughout G2, sister chromatid cohesion may prevent free break ends from long-range diffusion, thereby promoting post-replicational repair processes (40) until cohesin is unloaded at the onset of anaphase. Our data support a model in which radiation-induced DSBs are less diffusible in G2 cells due to sister chromatid cohesion, resulting in a reduced probability of end joining leading to the joining of incorrect break ends. This model is consistent with results from a recent study in our group which demonstrated enhanced restriction fragment reconstitution in highly condensed centromeric DNA in comparison to euchromatic DNA (19). It is worth noting that the G1 phase populations used in the present study were obtained by growing cells to confluency. Confluent, non-growing G1 cells may have exited the cell cycle and possibly could possess a state of chromatin condensation different from growing G1 cells. It is also interesting that G2 phase and non-growing G1 phase CHO cells show approximately the same level of radiosensitivity (24), consistent with our previous notion that DSB mis-rejoining may not necessarily be a lethal event and more likely may have consequences for other biological end-points such as mutations and chromosome exchange aberrations (41).

    ACKNOWLEDGEMENTS

    We would like to thank P. Jeggo for providing AA8 and V3 cells, R. Greinert for sending K1 cells, L. Thompson for providing irs1SF cells, A. Krempler and P. Jeggo for comments on the manuscript and M. Kühne for help in preparing figures. xrs-6 cells (European Collection of Cell Cultures) are commercially available. Financial support was provided by the Deutsche Forschungsgemeinschaft (grants Lo 677/1-1 and Lo 677/1-2) and the Radiation Protection Programme of the European Community (FIGH-CT-1999-00012).

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