Mutagenesis, Vol. 16, No. 3, 225-232,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines
MGC, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, PO Box 9503, 2300 RA Leiden, Wassenaarseweg 72,The Netherlands and 1 Center for Radiological Research, Columbia University, College of Physicians and Surgeons, 630W 168th Street, New York, NY 10032, USA
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Abstract |
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Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase
and 
, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy. The results indicate that exposure to X-rays triggers the induction of PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway. X-ray-induced repair synthesis was greatly inhibited by treatment of cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step. |
Introduction |
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Ionizing radiation (IR) induces a variety of lesions in cellular DNA. These include DNA base damage, single- and double-strand breaks and DNA–protein crosslinks (Teoule, 1987
). Cells use different repair pathways to remove this damage. DNA strand breaks are repaired either by non-homologous end joining or homologous recombination repair pathway and the oxidative base lesions generated by free radicals are repaired via the base excision repair (BER) pathway (Ward, 1988). The spectrum of DNA lesions and repair pathways induced by IR are different from those induced by UV irradiation and other genotoxic agents. The UV-induced pyrimidine dimers or 6-4 photoproducts are efficiently removed from cells by a mechanism known as nucleotide excision repair (NER). Many of the gene products involved in the NER process in mammalian cells have been identified through studies on inherited, sun-sensitive human autosomal recessive disease xeroderma pigmentosum (XP). XP includes seven complementation groups (A–G) and the proteins encoded by XP genes play important roles in damage recognition and incision steps of NER. In addition to XP factors, DNA polymerase 
and , proliferating cell nuclear antigen (PCNA), replication factor C, replication protein A and DNA ligase are also involved in the NER process (Sancar, 1994; Shivji et al., 1995). During the UV-induced NER process, the damaged DNA is often excised as an oligomer of ~30 nt. On the contrary, the length of the repair patches induced by IR is only 5–7 nt in length (Baide et al., 1998). Earlier studies have shown that both of these repair processes occur by different mechanisms involving a different sub-set of proteins (Digiuseppe et al., 1990; Seki et al., 1990). However, both NER and BER processes require PCNA as one of the major components (Muira et al., 1996; Krisna et al., 1994; Shivji et al., 1992, 1995).
PCNA is an auxiliary factor for the DNA polymerase and , and found to be important for DNA replication and repair. PCNA was initially thought to be a moving platform for DNA polymerase or but recent studies indicate that PCNA is involved in a variety of important cellular processes including cell cycle control, DNA replication and excision repair (Jonsson and Hubscher, 1997; Prosperi, 1997). In yeast and mammalian cells, three identical monomers of PCNA assemble into a toroidal ring that encircles the template as a `sliding camp' during DNA synthesis. This structure may serve as an anchor at the 3'-OH terminal of the nascent DNA strand, with one face of the ring bound to DNA polymerases (Amin and Holm, 1996; Kelman, 1997; Krisna et al., 1994). This trimeric form of PCNA is considered to be the functional form of PCNA, which enhances the processivity of polymerase .
Two pathways of BER in mammalian cells, long- and short-patch BER has been proposed (Frosina et al., 1996) and different polymerases are involved in these pathways (Fortini et al, 1998; Gray et al., 1999). Short-patch BER is a DNA polymerase ß-dependent pathway that requires an unaltered deoxyphosphate (dRP) sugar moiety as the AP site. For long-patch BER, flap endonuclease, FEN-1 removes the 5-terminal dRP moiety along with at least one adjacent nucleotide to leave a gap of two or more nucleotides (Kim et al., 1998; Klungland et al., 1997). The resynthesis for long-patch BER requires PCNA (Gray et al., 1999; Matsumoto et al., 1999). However, PCNA-independent and polymerase ß-mediated long-patch repair has also been reported (Prasad et al., 2000). Studies dealing with the assessment of the role of PCNA in the BER pathway in vivo are very limited as most of the repair studies were performed in vitro, i.e. removal of the damages from the naked DNA was monitored after incubation with cellular extracts.
In an effort to determine the involvement of PCNA in the X-ray-induced DNA repair pathway, we have used a quasi in vitro assay that enables labelling of the repair sites and PCNA under physiological salt concentrations in a native chromatin template. This assay involves the use of agarose embedded cells (Jackson et al., 1994), which are permeabilized, and the repair sites are labelled in vitro by biotin 16-dUTP and other deoxynucleotides. Induction of PCNA was detected by indirect immunolabelling. Similar to that observed for UV irradiation, X-rays induced a homogeneous punctuated pattern of PCNA distribution in the interphase nuclei of normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B) cells. Although these cells differ from each other in their NER capacity, no difference was observed between them either in repair sites or PCNA complex formation. The pattern of PCNA distribution closely resembled that of the repair sites detected by biotin incorporation. Efficient induction of PCNA as well as repair sites induced by X-rays in NER defective XP-A cells indicate that BER pathway is responsible for the observed phenomenon. Although treatment of cells with aphidicolin and cytosine arabinoside inhibited the repair synthesis, the intensity as well as the distribution of PCNA remained unchanged in all the three cell lines. The persistence of the PCNA complex in the absence of detectable repair resynthesis strongly suggests the requirement of PCNA for the BER process at a step preceding the repair resynthesis step.
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Materials and methods |
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Cells culture
The primary fibroblast cells (VH-25, normal; XP25RO, xeroderma pigmentosum group A; and CSIAN, Cockayne syndrome complementation group B) were used. All cells were cultured in F10 medium supplemented with 10% fetal bovine serum and antibiotics in 2.5% CO2 atmosphere. Cells were grown until confluent and maintained in that state for 3 days without changing the medium prior to irradiation.
X-irradiation
The cells were irradiated at room temperature using a Muller machine operated at 200 kV and 8 mA at a dose rate 2 Gy/min.
Repair inhibitors
The confluent cells were incubated with 50 µM aphidicolin or 0.1 mM 1-ß-D-arabinofuranisylcytosine (araC) 4 h before irradiation. In all the subsequent incubations these inhibitors were present at the same concentrations.
Encapsulation and permeabilization of the cells
Cells were encapsulated in agarose according to the method described by Jackson et al. (1994). Briefly, confluent cells were trypsinized and resuspended in phosphate-buffered saline (PBS) at a concentration 2x105 cells/ml. An equal volume of cell suspension in PBS was mixed gently with 1% low gelling agarose (Sigma) at 39°C. An equal volume of paraffin oil was mixed thoroughly with the cell suspension and kept on ice for 1 min with gentle swirling. The whole mixture was kept at room temperature for 5 min. Finally, they were centrifuged repeatedly to remove paraffin oil. The agarose beads were checked under a phase contrast microscope. Each bead contained approximately five to six cells. The beads were incubated in PBS for 1 h at 37°C before irradiation.
Irradiated or unirradiated beads containing cells were kept in conditioned medium for different times at 37°C. After incubation they were washed twice with PBS and three times with cold physiological buffer [PB: 100 mM KH2PO4, 130 mM KCl, 10 mM Na2PO4, 1 mM MgCl2, 1 mM Na2ATP (Sigma: type II), 1 mM dithiothreitol, pH 7.4]. The beads were then incubated with streptolysin O (SLO, Wellcome, 5 i.u./ml) on ice for 15 min and washed with cold PB to remove unbound SLO. The beads were then incubated for 1 min at 37°C and then washed twice with cold PB.
Labelling of repair patches
Irradiated or unirradiated agarose embedded permeabilized cells were then incubated with 10x repair mix containing 2.5 mM of each dGTP, dCTP, dATP and 100 µM Biotin-16-dUTP (Boehringer Mannheim), 50 mM phosphate buffer pH 7.4 and 2.5 mM MgCl2 for 15 min at 37°C. The reaction was stopped with cold PB and washed three times with cold PB. The beads were then incubated on ice with an equal volume of 0.5% Triton in PB for 10 min. After several washes the cells were fixed with 2% formaldehyde for 10 min at room temperature. Finally, the beads were incubated with 1% bovine serum albumin (BSA) and 1% goat serum (Jackson Laboratory) in PB for 30 min at room temperature to prevent non-specific binding of the antibodies. The beads were washed once with wash buffer (PB containing 0.05% Tween-20, and 0.1% BSA). The sites containing the biotin were detected using avidin-FITC (Sigma; 1:500 dilution) incubation at room temperature for 30 min followed by further incubation at room temperature for 30 min with goat anti-avidin (Sigma; 1:200 dilution), finally the beads were incubated with avidin-FITC again for 30 min at room temperature. After each incubation, the beads were washed three times with wash buffer. Beads (25 µl) were placed on a glass slide under a coverslip in Vectashield (Vector Labs). The slides were immediately viewed under a fluorescence microscope (Zeiss Auxioplan Microscope). Images were captured using a Nu200 CCD camera (Photometric) linked to an Apple power Macintosh computer. For the determination of fluorescence intensity, nuclei were selected randomly and the intensity was measured using IPLab Spectrum software. At least 30–50 nuclei were analysed for each point and average intensity was calculated in arbitrary units.
Immunofluorescence staining of PCNA
The procedure for PCNA immunostaining was essentially the same as described previously (Aboussekhra and Wood, 1995). The cells were grown on a coverslip for immunofluorescence, washed with PBS and irradiated with X-rays. After incubation, the cells were incubated with cold PBS containing 0.25% Triton for 15 min at 4°C. The coverslip was then washed with PBS and fixed with methanol for 15 min at –20°C. The fixed cells were then washed in PB and incubated with mouse monoclonal anti-PCNA antibody (Sigma; 1:100 dilution) for 30 min at room temperature. The cells were washed three times with PBS containing 0.05% Tween-20, followed by a further 30 min incubation at room temperature with donkey anti-mouse IGg conjugated with FITC (Jackson Laboratory; 1:100) in the dark. The coverslip was mounted in Vectashield (Vector Labs) and viewed under a fluorescence microscope. The images were captured and analysed as described before.
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Results |
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In this study, we have utilized a quasi in vitro assay to label the sites of repair and that of PCNA induced by X-rays. This assay involves the encapsulation of cells in agarose microbeads. The encapsulated cells are then permeabilized and the repair sites are detected using biotinylated 16-dUTP and dNTPs. This assay has been successfully used previously to map the repair and transcription sites in the interphase nuclei of human cells after UV damage (Aboussekhra and Wood, 1995
). This assay was used in the present study to label the repair sites induced by X-rays. In X-irradiated cells, PCNA was found homogeneously in a punctuated form throughout the interphase nuclei in all three cell lines (Figure 1A). Unirradiated control cells did not show any PCNA staining. The presence of the PCNA complex in XP-A cells after X-rays, but not after UV irradiation (Miura et al., 1992) suggests that PCNA foci formation is intimately associated with BER activity. Although CS-B cells have been shown to be sensitive to IR-induced DNA damage (Leadon and Copper, 1993), we did not observe any change in the level of both repair synthesis and PCNA distribution in these cells. The images of PCNA immunostaining and repair patches in X-irradiated (20 Gy) cells at different incubation times showed that the intensity of PCNA immunofluorescence was found to be time dependent in all three cell lines (Figure 1B). The sites of repair detected by avidin-FITC were very similar to PCNA distribution. In Figure 1C and D intensity of PCNA immunostaining and repair patches were plotted respectively, at different post-irradiation time for all the cell lines irradiated with 20 Gy of X-rays. The PCNA immunostaining and repair patches can be detected within 10 min of irradiation. The intensity of both PCNA and repair labelling reached a peak at 30 min after X-rays and then gradually declined. For each time point, 30–50 nuclei were analysed for the quantification of fluorescence intensity. The values were normalized to untreated control cells at each point.
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Images of PCNA immunostaining and repair patches with different doses in normal cells are presented in Figure 2A
. The intensity of both PCNA immunostaining and repair patches was found to increase with the dose. Similar experiments done for the other two NER defective cell lines (XP-A, CS-B) did not reveal any differences either in PCNA immunostaining or repair patches as compared with normal cells. The intensity of PCNA immunostaining and repair patches was plotted as a function of dose (Figure 2B and C). Accumulation of PCNA and induction of repair patches were similar for all the cell types as the intensity of both the PCNA and repair patches increased with dose. Similar dose-dependent increase of PCNA has also been observed in N'-methyl-N'-nitrosoguanine-treated cells (Savio et al., 1998).
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We next determined whether or not the recruitment of PCNA after X-rays is dependent on the repair resynthesis step. For this purpose, the potent drugs such as aphidicolin and araC at a concentration of 50 and 100 µM, respectively, were used. The cells were incubated with the two drugs for 4 h before irradiation, washed, X-irradiated (10 Gy) and subsequently incubated in the same conditioned medium containing aphidicolin and araC. The results of these experiments in normal cells indicated that the induction of repair patches was reduced but PCNA immunostaining remained more or less the same (Figure 3A
). These two drugs inactivate DNA polymerases , or thereby affecting the repair synthesis (Mizayans et al., 1992). Although repair inhibition was observed, intensity of PCNA immunostaining remained the same in all three cell lines. The fluorescence ratio for PCNA immunostaining was 3.2, 3.1 and 2.9 for 10 Gy of X-rays and 30 min of post-incubation in three cell lines, i.e. normal, XP-A and CS-B respectively. The values of PCNA fluorescence intensity in normal, XP-A and CS-B cells after treatment with aphidicolin and araC were found to be 3.1, 2.5, 2.9 and 2.9, 2.8, 2.75 respectively. In the same experiments, the repair values were 3.8, 4, 3.6 for irradiation alone and 1.5, 1.6, 1.4 for radiation with aphidicolin and 1.8, 1.9, 1.6 for radiation with araC in normal, XP-A, CS-B cells respectively (Figure 3C).
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Discussion |
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The involvement of PCNA in the resynthesis step of NER has been very well established by a number of studies (Aboussekhra and Wood, 1995
; Bravo and Macdonald-Bravo, 1985; Miura et al., 1992). IR-induced PCNA foci formation is efficient in NER-defective XP-A and transcription coupled repair (TCR)-defective CS-B cells. Similar distribution of repair and PCNA foci in the interphase nuclei of human cells suggests that the PCNA foci may be intimately connected with BER activity. The intensity of PCNA staining reached a peak 30 min after irradiation and the intensity declined during the longer post-incubation times. This time-dependent association of PCNA foci with nuclear substructures seems to correlate with the kinetics of BER activity.
Using a quasi in vitro assay, we were able to localize the repair sites induced by X-rays for the first time in intact chromatin preparations. The length of the repair patches induced by X-rays is known to be different from UV. IR-induced damages are removed from the genome with different kinetics. The fast and slow repairable damages are assumed to be base or sugar damage and single-stranded breaks respectively (Ward, 1988). We have labelled the cells for 15 min in the presence of biotin to visualize the patches, whereas UV-induced repair patches are easily detectable by 5 min of labelling after a dose of 2 J/m2 (unpublished observation). The difference could be due to the length of the repair patches generated after UV- and X-ray-induced damage. In XP-A cells, UV-induced repair patches as well as the PCNA immunostaining are absent. The detection of PCNA foci after X-rays in XP-A cells indicate that XP-A cells are proficient in IR-induced BER activity.
Cells from CS patients are generally sensitive to all kinds of DNA damaging agents (Friedberg, 1996). CS cells have been shown to be proficient in global genome repair of UV-induced damage but are unable to remove lesions from the transcribed strand of active genes known as `transcription coupled repair', which is yet another sub-pathway of NER. We have analysed CS-B cells in this study to determine whether they have any deficiency in repair induced by X-rays. Also, there is a report stating that CS cells are sensitive to IR (Leadon and Copper, 1993) and that they are defective in TCR removal of oxidized base lesions. In this study, we did not observe any deficiency in the intensity of PCNA staining and the biotin-labelled repair sites in XP-A and CS-B cell lines. As TCR removes the lesions from only 5% of the genome, its impact on the overall distribution of PCNA intensity is hard to detect with the assay we have employed in this study. We have observed that PCNA foci formation triggered by X-rays was not at all affected by treatment with inhibitors of DNA polymerase(s) involved in resynthesis step. It is not clear whether PCNA recruitment to the repair sites precedes the resynthesis step of BER. PCNA physically interacts with XP-G protein, an endonuclease involved in 3' incision of UV-damaged DNA during NER (Gary et al., 1997). This raises the possibility that PCNA could be a part of the complex in the BER process before the repair synthesis start and thus the inhibition of repair synthesis may not affect the PCNA immunostaining. A recent study has shown that PCNA binds to single-strand breaks with high affinity and may recruit chromatin assembly factor to maintain the integrity of higher order chromatin structure (Moggs et al., 2000). An alternative possibility is that PCNA may bind to the damaged sites thereby facilitating the repair of lesions by recruiting the repair factors involved in the BER pathway. Gray et al. (1999) also showed in an in vitro BER reconstituted assay that PCNA is required for the formation of excised reaction intermediates. Another possibility is that the X-rays produce a wide range of damage and the repair mechanism(s) of such damages may be independent of each other. As different sets of polymerases are involved in BER (Fortini et al., 1998), inhibition of specific polymerases could block any one of the repair processes, while other repair pathways, which require PCNA, may continue uninhibited. These suggestions are, however, purely speculative and require further studies to resolve.
It is generally believed that in UV-irradiated cells the formation of `repairosome' occurs in the vicinity of the damage for efficient removal of the damage (Svejstrup et al., 1995). In NER, PCNA was found to be an important component of this complex and the function of PCNA in NER may not be restricted to only the processivity of DNA polymerase for DNA synthesis (Jonsson and Hubscher, 1997). It has been shown that PCNA is redistributed from a soluble into a chromatin bound insoluble form following UV irradiation (Toschi and Bravo, 1988). In the case of X-rays, PCNA was found to be associated with DNA as PCNA immunostaining vanished completely when the cells were treated with DNase I. Recently it has been observed that the P53/P21 signal transduction pathway plays a significant role in the regulation of the PCNA response to IR (Wenz et al., 1998). In UV-irradiated cells, inhibition of DNA polymerase or is known to result in reduced repair labelling (Mirzayans et al., 1993). In UV-irradiated cells, it has been observed that the extent of UDS is comparable with their immunostaining of PCNA (Aboussekhra and Wood, 1995). The PCNA immunostaining was not detectable in XP-A and XP-G cells after UV irradiation. These two cell lines are also completely deficient in NER. These two proteins actively participate during the damage recognition/incision step of NER in normal cells. This shows that PCNA complex formation does not occur in the absence of damage recognition and incision steps of NER.
Unlike UV irradiation, we detected the PCNA staining after X-ray in XP-A cells even after the inhibition of repair synthesis by aphidicolin and araC. One major difference between UV and IR is that the DNA strand breaks are induced directly by IR while the strand breaks arise in UV-treated cells as repair intermediates after the incision reaction. The absence of DNA strand breaks in incision-defective XP-A and XP-G cells might explain the lack of PCNA complex formation in these cells after UV irradiation. In view of the high affinity of PCNA to DNA strand breaks (Moggs et al., 2000), it is logical to expect the rapid recruitment of PCNA to IR-induced strand breaks.
In this study, we have demonstrated the induction of PCNA after X-ray in NER-defective XP-A and TCR-defective CS-B cells. The dose- and time-dependent formation of repair sites and their close resemblance in distribution to PCNA sites in the nuclei indicate a role for PCNA in the BER pathway. We have also shown that the recruitment of PCNA to damaged sites occurs independently of the repair resynthesis step. It is, however, unclear whether the PCNA complex is a prerequisite for BER activity. Further studies are required to elucidate the precise role(s) of PCNA in the repair of DNA strand breaks and oxidative DNA base damages.
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Acknowledgments |
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We thank Jan Boie for his help with image analysis. This project was supported by an EU Grant to A.T.N.
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Notes |
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2 To whom correspondence should be addressed. Tel: +31 715276164; Fax: +31 715221615; Email: natarajan{at}lumc.nl
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References |
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Received on June 20, 2000; accepted on December 20, 2000.
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 E. Larsen, C. Gran, B. E. Saether, E. Seeberg, and A. Klungland Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst Stage Mol. Cell. Biol., August 1, 2003; 23(15): 5346 - 5353. [Abstract] [Full Text] [PDF]
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