Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2PCNA− mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.

This article has an associated First Person interview with the first author of the paper.

The extent of DNA damage and the efficiency of cells in repairing this damage are closely linked to several pathological processes. DNA lesions not properly repaired can lead to mutations or wider-scale genome aberrations that compromise cell or organism viability. To avoid this threat, cells have evolved a network of mechanisms to detect and repair a plethora of distinct genotoxic lesions (Jackson and Bartek, 2009). This network mainly consists of base excision repair (BER), which is able to repair single-stranded breaks and non-helix-distorting base alterations (Hegde et al., 2008), and nucleotide excision repair (NER), which is involved in removing different types of DNA-helix-distorting lesions (Gillet and Schärer, 2006; Marteijn et al., 2014). In addition, the repair network also involves homologous recombination (HR) and non-homologous end joining (NHEJ), employed by cells to repair double-strand breaks, and mismatch repair (MMR), which recognizes base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination (Jiricny, 2006).

Among these, the NER pathway is a highly versatile and complex system by which a large variety of DNA damage is eliminated from the genome. NER can be initiated by global genome repair (GG-NER), which occurs in the whole genome, or by transcription-coupled NER (TC-NER), which repairs lesions in the transcribed strand of active genes (Fousteri and Mullenders, 2008; Hanawalt, 2002). Hereditary NER defects cause autosomal recessive syndromes, such as xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome, all characterized by deficiencies in DNA repair (Niedernhofer et al., 2011). The former, which results in a strong predisposition to skin cancer, underlines the importance of the NER system for suppression of UV-induced mutagenesis and carcinogenesis (de Vries et al., 1995). Many studies have also linked DNA damage and repair efficiency with the susceptibility and progression of several types of cancer (Goode et al., 2002; Merolla et al., 2016; Ramos et al., 2004), and NER plays a critical role in the maintenance of genomic integrity because of its broad substrate specificity.

Given all of the above, any information concerning the efficiency of DNA repair processes, as well as the role of specific NER proteins and their partner interactions, is very important both for basic and clinical research. Over the years, several approaches have been developed to directly measure DNA repair synthesis (Figueroa-González and Pérez-Plasencia, 2017; Lehmann, 2011), starting from the classical unscheduled DNA synthesis (UDS) assay (Rasmussen and Painter, 1964), recently modified by replacing 3H thymidine with its analogue 5-ethynyluridine (EdU), thereby avoiding radioactivity (Limsirichaikul et al., 2009). In parallel, for studying NER mechanisms, different cell-free systems have been developed based on reconstitution of NER using recombinant proteins (Aboussekhra et al., 1995; Araújo et al., 2000; Garner and Costanzo, 2009; Mu et al., 1995; Wood et al., 1988), and, more recently, detection of NER factors at local damage sites by immunofluorescence, localized irradiation and chromatin immunoprecipitation techniques are the most used methods for monitoring the spatial organization of NER factors (Dinant et al., 2007; Dutto et al., 2017; Katsumi et al., 2001; Vermeulen, 2011).

Nevertheless, many of the above cell-free systems try to reproduce biochemical reactions on the damaged sites, sometimes containing specific lesions (Shivji et al., 2006), by using purified proteins and plasmid DNA (Aboussekhra et al., 1995; Biggerstaff and Wood, 1999). These approaches have some limitations; they can lack the complexity of chromatin organization in mammalian nuclei, involve difficult purification of large proteins and do not account for unknown proteins in the pathway, which cannot be reconstituted. Similarly, yeast- or other microorganism-based cell-free systems present significant disadvantages because some mammalian pathways involved in DNA damage responses are not represented (Hoeijmakers, 1993). In addition, Xenopus or mammalian extracts and nuclei, proposed so far as cell-free systems, preferentially assess several aspects of the DNA damage response, including DNA damage checkpoints, without addressing effectiveness of the DNA repair process (Costanzo and Gautier, 2004; Garner and Costanzo, 2009; Roper and Coverley, 2012).

In this report, we have modified a functional cell-free system to evaluate DNA repair efficiency using isolated nuclei and cell extracts from different human cell lines. This is an adaptation of a mammalian cell-free system used for studying the initiation of DNA replication in vitro (Krude, 2000; Krude et al., 1997) and for its validation we have used both physical and chemical agents able to cause different types of DNA lesions. In particular, cells were exposed to UV radiation, directly inducing 6–4 photoproducts (6–4PPs) or cyclobutane pyrimidine dimers (CPDs) (Sinha and Häder, 2002), lesions mainly removed through activation of the NER process (Aboussekhra and Wood, 1994; Sancar and Tang, 1993). The effects of DNA polymerases and DNA synthesis inhibitors, together with the lack of DNA repair response in NER-defective XPC cells, were used to verify the specificity of the method. In addition, the opportunity to apply this cell-free in vitro system to the study of several types of DNA damage, including single-strand and double-strand breaks (SSBs and DSBs) caused by ionizing radiation (IR), addition of alkyl groups to bases by chemical agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and DNA adducts resulting from treatment with the chemotherapeutic agent cisplatin, has also been explored.

Detection of dUTP incorporation in UV-irradiated HeLa and HaCaT nuclei

In order to produce a population of damaged nuclei and the corresponding cytosolic extracts for cell-free experiments, mimosine-arrested G1-phase HaCaT and HeLa cells (Fig. S1) were irradiated with UVC to activate the NER pathway and immediately processed for isolation of nuclei. We initially confirmed the arrest in G1 phase using a double labelling with cyclin D1 (also known as CCND1) and PCNA, markers of G1 and S phase, respectively. Most of the permeabilized cells were stained positive for cyclin D1 and not for PCNA (Fig. 1A), as expected, and more than 90% of the nuclei were in the G1 phase, as determined by flow cytometry analysis (Fig. 1B). We then investigated whether DNA repair synthesis could be efficiently detected by incorporation of biotin-16-UTP (dUTP) into DNA. Incubation of HaCaT nuclei from control or irradiated cells in the corresponding cytosolic extracts resulted in about 9% and 94%, respectively, of the nuclei incorporating dUTP in vitro (Fig. 1C,E). Analysis by confocal microscopy at higher magnification showed a different pattern of dUTP fluorescence signal (Fig. 1D) between control and UVC-irradiated nuclei. A small proportion of nuclei (∼2%) had labelling that resembled the replication foci typical of very early S phase (Maya-Mendoza et al., 2009), whereas most of the UVC-damaged nuclei (92%) appeared homogenous, with very small foci distributed throughout the nucleus (Fig. 1D,E). As expected, analysis of dUTP-positive nuclei by flow cytometry (Fig. 1F,G) strongly confirmed that the mean fluorescence intensity (MFI) of dUTP-positive nuclei was significantly higher in UVC-treated samples than in the control samples. Similar results were obtained in isolated HeLa nuclei (Fig. S2), in which the percentage of nuclei showing a residual DNA replication activity was limited to 7%. To confirm that DNA synthesized in vitro was due to a DNA repair process induced by UVC irradiation and not due to any DNA replication events initiated in vivo, HaCaT cells were incubated with BrdU in the last hour before preparing nuclei and cell extracts. As shown in Fig. 2A, S-phase contaminant nuclei were highlighted by BrdU labelling in live cells (red fluorescence), while DNA synthesis in vitro was labelled by dUTP-incorporation (green fluorescence); colocalization of both signals (yellow dots) confirms the ability of S-phase nuclei to efficiently elongate DNA in vitro (Fig. 2A) (Krude et al., 1997). In contrast, UVC-treated nuclei were labelled only by dUTP, demonstrating that DNA synthesis was initiated in vitro following UVC irradiation. To demonstrate that DNA repair synthesis of the irradiated HaCaT nuclei was specifically activated by UVC radiation, triggering the NER process, the XP14BR cell line, derived from an XP-C (xeroderma pigmentosum group C) patient, was used as negative control. As expected, no difference in dUTP signal was observed in G1-arrested nuclei isolated from control and UVC-treated XP14BR cells, except for some DNA-replicating nuclei present only in the control sample (Fig. 2B). The absence of repair-related dUTP incorporation was also confirmed by flow cytometry analysis of both samples (Fig. 2C).

Fig. 1.

Biotin-d-UTP in vitro incorporation inisolated G1-phase nuclei from irradiated (UVC) and untreated (Control) HaCaT cells. (A) Representative fluorescence images of HaCaT cells synchronized in G1 and immunostained with anti-cyclin D1 (green fluorescence) and anti-PCNA (red fluorescence) antibodies, as markers of G1 and S phase, respectively. DNA was stained with Hoechst 33258 (blue fluorescence). (B) Flow cytometry analysis of the cell cycle distribution of HaCaT nuclei isolated from cells synchronized in G1, and the corresponding percentage of cells in each phase. (C) Representative images of in vitro DNA neo-synthesis after 2 h of incubation in standard conditions, as visualized by streptavidin–Alexa Fluor 488 (dUTP, green) and Hoechst 33258 (DNA, blue). (D) Higher magnification of replicative and repair patterns, as labelled by streptavidin–Alexa Fluor 488. (E) Visual scoring of nuclei for replicative and repair synthesis, expressed as positive percentage (%). (F) Flow cytometry quantification of repair synthesis expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (G) Monoparametric analysis of DNA repair synthesis by flow cytometry of samples marked with streptavidin–Alexa Fluor 488 (green fluorescence). Data from unlabelled, untreated cells are also shown (Blank). Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bars: 10 µM. Student's t-test; ***P<0.001.

Fig. 1.

Biotin-d-UTP in vitro incorporation inisolated G1-phase nuclei from irradiated (UVC) and untreated (Control) HaCaT cells. (A) Representative fluorescence images of HaCaT cells synchronized in G1 and immunostained with anti-cyclin D1 (green fluorescence) and anti-PCNA (red fluorescence) antibodies, as markers of G1 and S phase, respectively. DNA was stained with Hoechst 33258 (blue fluorescence). (B) Flow cytometry analysis of the cell cycle distribution of HaCaT nuclei isolated from cells synchronized in G1, and the corresponding percentage of cells in each phase. (C) Representative images of in vitro DNA neo-synthesis after 2 h of incubation in standard conditions, as visualized by streptavidin–Alexa Fluor 488 (dUTP, green) and Hoechst 33258 (DNA, blue). (D) Higher magnification of replicative and repair patterns, as labelled by streptavidin–Alexa Fluor 488. (E) Visual scoring of nuclei for replicative and repair synthesis, expressed as positive percentage (%). (F) Flow cytometry quantification of repair synthesis expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (G) Monoparametric analysis of DNA repair synthesis by flow cytometry of samples marked with streptavidin–Alexa Fluor 488 (green fluorescence). Data from unlabelled, untreated cells are also shown (Blank). Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bars: 10 µM. Student's t-test; ***P<0.001.

Fig. 2.

DNA repair specificity and kinetics. (A) Identification by confocal microscopy of G1 nuclei incorporating BrdU in HaCaT cells and dUTP in vitro for both replicating (control) and repairing (UVC) HaCaT nuclei. Sites of DNA synthesis in vivo (BrdU, red) and in vitro (dUTP, green) colocalized in the same nucleus (merge, yellow dots). DNA counterstaining with Hoechst 33258 dye is shown (DNA, blue). (B) Representative images of nuclei isolated from control and UVC-irradiated XP14BR cells. After 2 h incubation in standard conditions, a typical DNA replication pattern, observed only in the control sample, is revealed by streptavidin–Alexa Fluor 488 (dUTP, green) and Hoechst 33258 dye (DNA, blue). (C) Flow cytometry quantification of dUTP incorporation in XP14BR isolated nuclei expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (D) Timecourse of dUTP incorporation in UVC-exposed (40 J/m2) and control HaCaT nuclei harvested and fixed after incubation for the indicated times. The number of dUTP-positive nuclei is expressed as a percentage (%) or (E) quantified as MFI by flow cytometry analysis. (F) Incorporation of dUTP in nuclei isolated from HaCaT cells exposed to different doses of UVC, as indicated, or not exposed to UVC (control). The number of dUTP-positive nuclei is expressed as percentage (%) or (G) quantified as MFI by flow cytometry analysis. Data reported are mean±s.d. obtained from ≥3 independent experiments. The dUTP incorporation in the UVC-treated samples in D and F is highly significant (P<0.001) compared to the incorporation by control samples. Student's t-test; *P<0.05, **P<0.01. Scale bars: 10 µM.

Fig. 2.

DNA repair specificity and kinetics. (A) Identification by confocal microscopy of G1 nuclei incorporating BrdU in HaCaT cells and dUTP in vitro for both replicating (control) and repairing (UVC) HaCaT nuclei. Sites of DNA synthesis in vivo (BrdU, red) and in vitro (dUTP, green) colocalized in the same nucleus (merge, yellow dots). DNA counterstaining with Hoechst 33258 dye is shown (DNA, blue). (B) Representative images of nuclei isolated from control and UVC-irradiated XP14BR cells. After 2 h incubation in standard conditions, a typical DNA replication pattern, observed only in the control sample, is revealed by streptavidin–Alexa Fluor 488 (dUTP, green) and Hoechst 33258 dye (DNA, blue). (C) Flow cytometry quantification of dUTP incorporation in XP14BR isolated nuclei expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (D) Timecourse of dUTP incorporation in UVC-exposed (40 J/m2) and control HaCaT nuclei harvested and fixed after incubation for the indicated times. The number of dUTP-positive nuclei is expressed as a percentage (%) or (E) quantified as MFI by flow cytometry analysis. (F) Incorporation of dUTP in nuclei isolated from HaCaT cells exposed to different doses of UVC, as indicated, or not exposed to UVC (control). The number of dUTP-positive nuclei is expressed as percentage (%) or (G) quantified as MFI by flow cytometry analysis. Data reported are mean±s.d. obtained from ≥3 independent experiments. The dUTP incorporation in the UVC-treated samples in D and F is highly significant (P<0.001) compared to the incorporation by control samples. Student's t-test; *P<0.05, **P<0.01. Scale bars: 10 µM.

Fig. 2D and E show a timecourse of DNA repair in vitro as determined by fluorescence microscopy and flow cytometry analyses. An efficient and highly significant (P<0.001) DNA repair synthesis (∼85% of the nuclei) was already triggered by HaCaT UVC-irradiated cytosol at 10 min after starting the reaction. During the following time points, the percentage of DNA-repairing nuclei remained similar, oscillating in the range 82–90% (Fig. 2D). But, importantly, the quantification of dUTP fluorescence demonstrated that DNA repair synthesis increased significantly in a time-dependent manner, reaching the highest efficiency at 120 min of incubation (Fig. 2E); in fact, a slight but significant decrease in dUTP incorporation was measured at 240 min. Similarly, we observed a dose-dependent response, better highlighted as the MFI of the incorporated dUTP, which reached a maximum signal at 40 J/m2 (Fig. 2F,G). This dose was chosen for all the subsequent experiments to maintain the high sensitivity of the assay. In samples isolated from control cells, neither the number of dUTP-positive nuclei, nor their MFI, was significantly modified with time, indicating that these signals were mainly dependent on S-phase contaminants (Fig. 2D,E).

Incubation of UV-irradiated nuclei in cytosolic extract from control cells resulted in a reduction of 30% in the number of nuclei incorporating dUTP in vitro, whereas the addition of the buffer SuNaSp to the reaction resulted in a significant loss of the dUTP signal (Fig. S3A). A similar decrease was observed in UVC dose-effect and timecourse experiments using cytosol from control cells, as compared to nuclei incubated with UV-treated cytosol (Fig. S3B,C). These results demonstrated that activated repair factors in UV-treated cytosol are required to efficiently trigger the NER process.

Sensitivity of the in vitro NER cell-free system to DNA synthesis inhibitors

In order to further validate the detection of DNA synthesis triggered by NER activation and to assess the sensitivity of the cell-free assay, UVC-damaged nuclei in the corresponding cytosol were exposed to aphidicolin (Aph) and cytosine arabinoside (Ara-C), two powerful inhibitors of DNA replication, albeit by different mechanisms. The former blocks B-family polymerase activity, and the latter is incorporated into DNA instead of dCTP, primarily resulting in chain termination of DNA synthesis and inhibition of pol α. As shown in Fig. 3A,B, DNA repair synthesis was significantly prevented by each inhibitor. A decrease in the percentage of repairing nuclei of ∼30% and ∼12% was observed for Aph- and Ara-C-treated samples, respectively. When both inhibitors were used in combination, a greater percentage decrease of 57% was observed. Notably, most of the dUTP-positive nuclei, through visual inspection, showed a marked reduction in fluorescence signal, which was confirmed by flow cytometry analysis (Fig. 3C); MFI was further reduced by 70, 39 and 78% in samples treated with Aph, Ara-C and both Aph and Ara-C, respectively, when compared to UVC-irradiated nuclei.

Fig. 3.

In vitro DNA repair assay efficiency in HaCaT nuclei incubated with 1 mM Aph and 20 µM Ara-C. (A) Representative fluorescence images of UVC-treated nuclei incubated in standard conditions and marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). Aph and Ara-C treatments are indicated. (B) Visual scoring of DNA repair synthesis of nuclei, expressed as positive percentage (%). (C) Flow cytometry quantification of DNA repair synthesis expressed as arbitrary units of the mean fluorescence intensity (MFI a.u.). Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bar: 10 µM. Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.

Fig. 3.

In vitro DNA repair assay efficiency in HaCaT nuclei incubated with 1 mM Aph and 20 µM Ara-C. (A) Representative fluorescence images of UVC-treated nuclei incubated in standard conditions and marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). Aph and Ara-C treatments are indicated. (B) Visual scoring of DNA repair synthesis of nuclei, expressed as positive percentage (%). (C) Flow cytometry quantification of DNA repair synthesis expressed as arbitrary units of the mean fluorescence intensity (MFI a.u.). Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bar: 10 µM. Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.

The NER cell-free system and exogenous proteins

In parallel, to assess the potential application of this system for studying mechanisms, dynamics and regulation of the DNA repair (NER) process, purified recombinant proteins known to be involved in NER, such as PCNA, DDB1 and DDB2, were used. Each protein was incubated with isolated nuclei for 10 min before adding the activated extract from UVC-treated cells. Among them, only PCNA caused a significant increase in dUTP incorporation (Fig. 4A,B) compared to the incorporation in UVC-treated extract alone and UVC-treated extract with BSA samples, the latter used as negative control. A slight increase, although not significant, was also noted in the presence of DDB2 protein (Fig. 4A,B). DDB2 is involved in the early stages of recognition of damage caused by UV radiation, thus contributing to the early recruitment of the GG-NER proteins. In order to evaluate whether this result was dependent on competition with the endogenous protein, western blot analysis was performed in both cell lines used in this study. The results showed that in HaCaT cells, DDB2 levels were very high in the nuclear compartment, in contrast to HeLa cell nuclei, in which the DDB2 band was barely detectable (Fig. 4C). For this reason, HeLa nuclei were chosen to further investigate whether DDB2 influences the NER process in vitro, together with a mutant form unable to interact with PCNA (DDB2PCNA−), which has been previously demonstrated to delay the removal of UV-induced DNA damage (Cazzalini et al., 2014; Perucca et al., 2018). Similar to the effects in HaCaT nuclei, addition of recombinant wild-type DDB2 protein (DDB2wt) did not affect DNA repair synthesis. However, a decrease in dUTP incorporation was clearly detectable in the presence of the DDB2PCNA− mutant protein (Fig. 4E); this lower efficiency in DNA repair is not due to a different amount of the proteins being recruited into the chromatin, as chromatin-bound levels appear to be comparable (Fig. 4D).

Fig. 4.

In vitro DNA repair assay in UVC-treated nuclei incubated with recombinant proteins involved in NER. (A) Representative images of UVC-treated HaCaT nuclei incubated with 400 ng of PCNA–His, 150 ng of DDB2wt–His or 150 ng of DDB1–His recombinant proteins, or 1 µg of BSA as a negative control. Nuclei were marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). (B) Flow cytometry analysis of UVC HaCaT nuclei incubated with recombinant proteins or BSA, as in A. DNA repair synthesis efficiency was expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (C) Western blot analysis of endogenous DDB2 levels detected in isolated nuclei and cytosolic extract of HeLa and HaCaT UVC-irradiated cells. (D) The chromatin-bound form of exogenous DDB2wt–His and DDB2PCNA−–His after the in vitro DNA repair assay, as analysed by western blotting using anti-histidine antibody. Histone H3 is shown as a loading control. (E) Flow cytometric analysis of UVC-treated HeLa nuclei incubated with 150 ng of DDB2wt–His or 150 ng of DDB2PCNA−–His recombinant proteins. DNA repair synthesis efficiency was expressed as MFI. Data reported are mean±s.d. obtained from ≥3 independent experiments. Blots in C, D are representative of ≥3 independent experiments. Scale bar: 10 µM. Student's t-test; *P<0.05, **P<0.01, ***P<0.001.

Fig. 4.

In vitro DNA repair assay in UVC-treated nuclei incubated with recombinant proteins involved in NER. (A) Representative images of UVC-treated HaCaT nuclei incubated with 400 ng of PCNA–His, 150 ng of DDB2wt–His or 150 ng of DDB1–His recombinant proteins, or 1 µg of BSA as a negative control. Nuclei were marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). (B) Flow cytometry analysis of UVC HaCaT nuclei incubated with recombinant proteins or BSA, as in A. DNA repair synthesis efficiency was expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (C) Western blot analysis of endogenous DDB2 levels detected in isolated nuclei and cytosolic extract of HeLa and HaCaT UVC-irradiated cells. (D) The chromatin-bound form of exogenous DDB2wt–His and DDB2PCNA−–His after the in vitro DNA repair assay, as analysed by western blotting using anti-histidine antibody. Histone H3 is shown as a loading control. (E) Flow cytometric analysis of UVC-treated HeLa nuclei incubated with 150 ng of DDB2wt–His or 150 ng of DDB2PCNA−–His recombinant proteins. DNA repair synthesis efficiency was expressed as MFI. Data reported are mean±s.d. obtained from ≥3 independent experiments. Blots in C, D are representative of ≥3 independent experiments. Scale bar: 10 µM. Student's t-test; *P<0.05, **P<0.01, ***P<0.001.

To further investigate the PCNA-dependent enhancement in dUTP incorporation, the exogenous recombinant protein was added to nuclei from UVC-treated cells incubated with cytosol from control cells and UVC-treated cells. The addition of recombinant PCNA promoted dUTP incorporation in UVC-irradiated nuclei when they were incubated with cytosol from untreated cells (Fig. 5A). In contrast, incubating irradiated nuclei with UVC-treated cytosolic extract depleted of endogenous PCNA (D.E.) resulted in a significant reduction in dUTP incorporation, whereas the addition of exogenous PCNA to D.E. restored the DNA repair capacity (Fig. 5B). Fig. 5C shows the high level of PCNA depletion attained after two rounds of incubation with p21–GST beads (lane 3). Because PCNA depletion in the cytosol (D.E.) did not completely abrogate dUTP incorporation, we asked whether the remaining repair activity of nuclei in D.E. extract could be dependent on the presence of some nucleosolic PCNA. In fact, endogenous PCNA was still present in UVC-treated nuclei (Fig. 5D, permeabilized −). Therefore, to eliminate this residual PCNA, nuclei were permeabilized to allow a complete release (Fig. 5D, permeabilized +). After this procedure, a significant reduction in dUTP incorporation was found in the permeabilized UVC sample (Fig. 5E), as compared to unpermeabilized nuclei, probably due to the release of other specific DNA repair factors. However, a further significant reduction of DNA repair was observed in the presence of D.E. extract, which was complemented to some extent by adding back recombinant PCNA.

Fig. 5.

In vitro DNA repair assay in UVC-treated nuclei (intact or permeabilized) incubated with PCNA recombinant protein. (A) Flow cytometry analysis of UVC-treated HaCaT nuclei incubated with 400 ng of PCNA–His recombinant protein added to cytosolic extracts from control cells (cyt Control) or cytosol from UVC-treated cells (cyt UVC), compared to incubation with cytosolic extract from UVC-treated cells alone, alongside data from untreated nuclei with untreated extract (Control). DNA repair synthesis efficiency was expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (B) Flow cytometry analysis of UVC-treated HaCaT nuclei after incubation with PCNA-depleted extract (D.E.) or D.E. containing 400 ng of PCNA–His (D.E.+PCNA), compared to incubation with undepleted extract (UVC), alongside data from untreated nuclei with untreated extract (Control). (C) Western blot showing PCNA levels in UVC-treated cytosol (lane 1) and after one (lane 2) or two (lane 3) rounds of incubation with p21C–GST peptide bound to GSH beads, for PCNA depletion. (D) Loading of isolated intact nuclei from UVC-treated cells (−) and after permeabilization of nuclear membrane (+). H3 indicates histone H3 used as loading control. (E) Flow cytometry analysis of UVC-treated HaCaT permeabilized nuclei incubated with cytosol extract from UVC-treated cells (UVC) or PCNA-depleted extract (D.E.), or with D.E. plus 400 ng of PCNA–His. Control and UVC samples represent our cell-free test standard conditions (nuclei isolated from untreated cells or UVC-treated cells, respectively, incubated with the corresponding cytosol). Data reported are mean±s.d. obtained from three independent experiments. Blots shown in C,D are representative of three independent experiments. Student's t-test; *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

In vitro DNA repair assay in UVC-treated nuclei (intact or permeabilized) incubated with PCNA recombinant protein. (A) Flow cytometry analysis of UVC-treated HaCaT nuclei incubated with 400 ng of PCNA–His recombinant protein added to cytosolic extracts from control cells (cyt Control) or cytosol from UVC-treated cells (cyt UVC), compared to incubation with cytosolic extract from UVC-treated cells alone, alongside data from untreated nuclei with untreated extract (Control). DNA repair synthesis efficiency was expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (B) Flow cytometry analysis of UVC-treated HaCaT nuclei after incubation with PCNA-depleted extract (D.E.) or D.E. containing 400 ng of PCNA–His (D.E.+PCNA), compared to incubation with undepleted extract (UVC), alongside data from untreated nuclei with untreated extract (Control). (C) Western blot showing PCNA levels in UVC-treated cytosol (lane 1) and after one (lane 2) or two (lane 3) rounds of incubation with p21C–GST peptide bound to GSH beads, for PCNA depletion. (D) Loading of isolated intact nuclei from UVC-treated cells (−) and after permeabilization of nuclear membrane (+). H3 indicates histone H3 used as loading control. (E) Flow cytometry analysis of UVC-treated HaCaT permeabilized nuclei incubated with cytosol extract from UVC-treated cells (UVC) or PCNA-depleted extract (D.E.), or with D.E. plus 400 ng of PCNA–His. Control and UVC samples represent our cell-free test standard conditions (nuclei isolated from untreated cells or UVC-treated cells, respectively, incubated with the corresponding cytosol). Data reported are mean±s.d. obtained from three independent experiments. Blots shown in C,D are representative of three independent experiments. Student's t-test; *P<0.05, **P<0.01, ***P<0.001.

Sensitivity of the cell-free system to detect different DNA repair systems

Having verified the ability of the cell-free system to properly reveal UV-activated NER processes, we next investigated whether this system could be used to test the activity of different DNA repair systems. To this end, nuclei were isolated from cells exposed to different DNA-damaging agents, known to activate BER or SSB and DSB repair.

HaCaT cells were exposed to the oxidizing agent potassium bromate (KBrO3, 40 mM for 15 min), the alkylating MNNG (50 µM for 30 min), the DNA-cross-linking drug cisplatin (50 µM for 1 h) or X-irradiation (1 Gy). Induction of DNA damage was confirmed by the phosphorylation of histone H2AX (γH2AX; Fig. 6A,B), which was significantly activated in all nuclei isolated from treated cells, except for those exposed to X-rays. Nevertheless, nuclei isolated from X-irradiated cells stained positive for Ku80, a protein playing an important role in NHEJ, the main pathway activated in G1 phase after treatment with ionizing radiation (Fig. S4A). A significant DNA repair synthesis response, in terms of dUTP incorporation, was clearly evident in the all nuclei treated with MNNG or cisplatin, compared to incorporation in the untreated control nuclei (Fig. 6C,D). Similarly, a marked dUTP incorporation was induced by X-irradiation, although not all nuclei were responsive. By contrast, the level of dUTP incorporation strongly dropped in the KBrO3-treated sample, as shown by the low value of MFI evaluated by flow cytometry (Fig. 6C). Nevertheless, when the nuclei were observed by confocal analysis, many small foci appear distributed in the chromatin of KBrO3-treated nuclei. These foci are better visualized at higher magnification (Fig. 6E), whereby a different nuclear staining pattern and fluorescence intensity was visible among the nuclei damaged with the different genotoxic agents.

Fig. 6.

In vitro DNA repair assay after exposure to different DNA-damaging agents. Nuclei were isolated from HaCaT cells either untreated or treated with UVC (UV), 40 mM KBrO3, 50 µM MNNG, 50 µM cisplatin or 1 Gy IR (RX), respectively. (A,B) Detection by western blot and quantification analysis of the γH2AX response in HaCaT cells untreated or treated with the different agents, as indicated. The levels of H2AX were used as a loading control. (C) Flow cytometry quantification of DNA repair synthesis following treatment with the different agents, as indicated, expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (D) Representative images of nuclei incubated in standard conditions, with or without treatment with the different agents, and marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). (E) Higher magnification of DNA repair patterns in nuclei treated with the DNA-damaging agents, as indicated. Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bars: 10 µM. Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.

Fig. 6.

In vitro DNA repair assay after exposure to different DNA-damaging agents. Nuclei were isolated from HaCaT cells either untreated or treated with UVC (UV), 40 mM KBrO3, 50 µM MNNG, 50 µM cisplatin or 1 Gy IR (RX), respectively. (A,B) Detection by western blot and quantification analysis of the γH2AX response in HaCaT cells untreated or treated with the different agents, as indicated. The levels of H2AX were used as a loading control. (C) Flow cytometry quantification of DNA repair synthesis following treatment with the different agents, as indicated, expressed as arbitrary units (a.u.) of the mean fluorescence intensity (MFI). (D) Representative images of nuclei incubated in standard conditions, with or without treatment with the different agents, and marked with streptavidin–Alexa Fluor 488 (green) and Hoechst 33258 dye (blue). (E) Higher magnification of DNA repair patterns in nuclei treated with the DNA-damaging agents, as indicated. Data reported are mean±s.d. obtained from ≥3 independent experiments. Scale bars: 10 µM. Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.

In this study we have used isolated nuclei, and their cytosolic extracts, from G1-synchronized cells exposed to DNA-damaging agents to assess the ability of a human cell-free system to carry out DNA repair synthesis. The results provide evidence that this cell-free approach can be exploited as an in vitro assay for investigating DNA repair activity. Specificity of the damage response in this nucleus-based model was demonstrated by the absence of dUTP signals in nuclei incubated in buffer, by the significant inhibition of DNA synthesis in the presence of Aph and Ara-C, either alone or in combination (Crute et al., 1986; Huberman, 1981; Mirzayans et al., 1994), and especially by the absence of a DNA repair response in G1 nuclei isolated from NER-defective XPC14BR cells. Moreover, the high percentage (98%) of dUTP-positive G1 nuclei, relative to unirradiated samples, as well as the absence of BrdU incorporation in living cells before nuclei isolation indicated that DNA synthesis initiated in vitro in UVC-exposed nuclei is due to DNA repair and not to initiation of DNA replication. This evidence is consistent with the observation that dUTP incorporation occurred throughout the chromatin, as both a uniformly distributed signal and a large number of very small foci, different from the typical pattern previously observed for early, mid and late S phase replication (Hozák et al., 1994; Pierzyńska-Mach et al., 2016).

The use of cell-free systems to study DNA repair is not a new concept. However, previous studies have used ‘naked’ damaged DNA substrates, generally of small size, in the presence of purified or recombinant NER proteins (Aboussekhra et al., 1995; Hansson et al., 1990; Lehmann, 2011; Mu et al., 1995; Zirkin et al., 2014). Although these systems enabled advancements in our understanding of the mechanism of NER, they do not provide information on DNA repair mechanisms in the context of chromatin in the whole nucleus. In living cells, genomic DNA is huge and organized in chromatin structures, which may play essential roles in the dynamics of repair processes.

Osmotic disruption of confluent cultured human fibroblasts has also been used as cell-free model for the specific measurement of repair DNA synthesis by using the nucleoside [3H]dTMP as a precursor (Ciarrocchi and Linn, 1978; Kaufmann et al., 1983). The intrinsic features of the experimental approach described here provide some clear advantages. As demonstrated, our approach represents a functional, non-radioactive, simple and reproducible technique to assess the DNA repair process in functionally and structurally intact nuclei and in the presence of damage-activated extracts. The dUTP-labelled DNA is easily visualized by fluorescence microscopy and quantified both by scoring dUTP-positive nuclei and measuring immunofluorescence intensity by flow cytometry. Another advantage of this procedure is its responsiveness to different types of DNA lesions. DNA damage induced in intact cells by oxidation, alkylation, treatment with cross-linking agents or radiation exposure trigger specific DNA repair pathways in isolated nuclei, compatible with the different patterns of foci distribution and size observed in each sample. In particular, a stronger dUTP signal was observed in nuclei isolated from cells treated with cisplatin or MNNG, the former essentially activating the NER pathway, the latter inducing BER in our experimental conditions. Repair of damage induced by MNNG treatment has been shown to occur primarily through the short-patch BER system via a DNA polymerase β (pol β)-dependent reaction (Kim and Wilson, 2012); however, the strong signal of dUTP incorporation into nuclei suggests that repair of MNNG-induced DNA adducts likely takes place via removal and insertion of 20–25 nucleotides (long-patch), involving PCNA-dependent polymerase, as we have previously demonstrated (Stivala et al., 1993). This observation is supported by the very low signal of DNA repair synthesis after treatment with the oxidative agent KBrO3. In fact, we observed only a few nuclei to be weakly dUTP-positive following KBrO3 treatment, suggesting that fewer dUTP incorporation events were triggered by a short-patch BER system, compared to incorporation following NER. Detectable dUTP incorporation, although to a lesser extent than seen after MNNG and cisplatin treatment, was produced by X-irradiation. The DNA repair pattern following X-irradiation differed from those obtained following treatment with the other agents, including KBrO3, by not being randomly distributed and by tending to occur in clusters; this observation agrees with observations of lesions termed multiply damaged sites, which have been already reported (Wallace, 1998; Ward, 1988). The DNA damage caused by irradiation is dependent on both direct effects and indirect effects, the latter associated with the generation of reactive oxygen species (Aparicio et al., 2014), which induce mainly SSBs and DSBs, although oxidized base damage is also known to occur (Illner and Scherthan, 2013). Considering HR repair occurs in the S and G2 phases, the pattern of dUTP foci detected in G1-arrested nuclei should be dependent on NHEJ or alternative end-joining (A-EJ) repair processes, along with BER. Colocalization of dUTP sites with XRRC1 (X-ray repair cross-complementing protein 1) (Fig. S4) strongly supports the involvement of the BER sub-pathways, in which XRCC1 and PARP [poly(ADP-ribose) polymerase] are principal players, but repair via Al-EJ or NHEJ cannot be excluded (Zhao et al., 2019). It is not clear, however, why not all nuclei are positive for dUTP, given that the DNA damage occurred to all nuclei, as assessed by a Comet test (data not shown) and by the binding of Ku80 to damaged DNA. Further studies may be envisaged in order to elucidate this issue.

Another significant advantage of our procedure is the possibility to introduce or deplete recombinant wild-type or mutated proteins into the extract, therefore enabling investigation of their roles in DNA repair. In UVC-irradiated nuclei from HaCaT cells, PCNA showed a significant positive influence on DNA repair synthesis; furthermore, its addition as exogenous recombinant protein complemented the reduction in DNA repair observed in nuclei (both intact or permeabilized) incubated in PCNA-depleted cytosol. Conversely, addition of DDB2, known for its role in NER after UV-induced DNA damage, slightly increased dUTP incorporation, although not significantly. One possible explanation for this result is competition between the exogenous and endogenous DDB2 protein, considering the high amount of endogenous DDB2 in HaCaT nuclei. In fact, using HeLa nuclei, in which nuclear endogenous DDB2 is only weakly detectable, we observed an increase in the repair efficiency in the presence of recombinant DDB2wt protein. An additional difference in the efficiency of UDS was detected using a DDB2 mutant protein unable to interact with PCNA (DDB2PCNA). Consistent with our data previously published, a significant reduction in dUTP fluorescence was measured, confirming the delayed removal of UV-induced DNA damage by this mutated form (Cazzalini et al., 2014; Perucca et al., 2018). We are currently trying to improve the sensitivity of this cell-free system by using biochemical knockouts for future studies to evaluate whether, and by which mechanism, a given protein or protein complex is implicated in DNA repair.

Cell cultures and synchronization

The human keratinocyte cell line (HaCaT, catalogue code: BS CL 168) was purchased from IZSLER (Italy). HeLa S3 cell line (HeLa, catalogue code: 87110901) was purchased from ECACC (European Collection of Authenticated Cell Cultures). HaCaT and HeLa cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Life Technologies-Gibco), 2 mM L-glutamine (Life Technologies-Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO2 atmosphere. XP14BR, a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C, and immortalized with pSV3neo vector, were kindly provided by Alan R. Lehmann (University of Sussex, Brighton, UK) (Arlett et al., 2006; Chavanne et al., 2000). They were cultured in DMEM supplemented as above. All cell lines were periodically tested for mycoplasma contamination.

To prepare G1-phase nuclei and cytosolic extracts, cells were synchronized by serum starvation (0.5% FBS) for 3 d, then arrested in late G1 phase by adding 0.5 mM L-mimosine (Sigma) to serum-starved cells for 24 h (Krude, 2000). Cell cycle synchronization was verified by BrdU incorporation (20 µM, Sigma) and flow cytometry analysis (PAS-II, Partec and Attune NxT, ThermoFisher). Synchronized HaCaT cells were also cultured on coverslips, then washed twice in PBS, and lysed for 10 min at 4°C in hypotonic buffer: 10 mM Tris-HCl (pH 7.4), 2.5 mM MgCl2, 0.1% Nonidet NP-40 and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). The samples were then washed in PBS, fixed in 4% formaldehyde for 5 min at room temperature and then post-fixed in 70% ethanol. After rehydration, samples were blocked in PBST buffer (PBS, 0.2% Tween-20) containing 1% bovine serum albumin (BSA), and then incubated for 1 h with specific monoclonal antibodies: anti-PCNA (1:100, Dako; RRID: AB_2160651) and anti-cyclin D1 (1:100, Santa Cruz; RRID: AB_2070436).

After three washes with PBST buffer, coverslips were incubated for 30 min with anti-mouse and anti-rabbit antibodies labelled with Alexa Fluor 594 or 488 (Molecular Probes), respectively. After immunoreactions, cells were incubated with Hoechst 33258 dye (0.5 µg/ml) for 2 min at room temperature and washed in PBS. Slides were mounted in Mowiol (Calbiochem) containing 0.25% 1,4-diazabicyclo-[2,2,2]-octane (Sigma-Aldrich) as antifading agent. All the fluorescent images of fixed cells and nuclei were taken with TCS SP5 II or TCS SP8 confocal microscopes, Leica, at 0.3 µm z intervals, using a 63× oil immersion objective.

Cellular treatments

HaCaT cells were exposed to different agents to induce DNA damage. UV-C exposure was performed using a lamp (Philips TUV-9) emitting mainly at 254 nm, at a dose of 10, 20 and 40 J/m2, as measured with a DCRX radiometer (Spectronics). X-ray irradiation was performed with a 6 MV LINAC (Varian) at IRCCS Maugeri (Pavia, Italy); cells were exposed to a dose rate of 1 Gy with a dose rate of 3 Gy/min at room temperature. Cisplatinum (cisplatin, 50 µM; Teva Pharma, Milan, Italy) or 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG, 50 µM; Sigma-Aldrich) were added to culture medium for 1 h or 30 min, respectively. Cell treatment with potassium bromate (KBrO3, 40 mM; Sigma-Aldrich) was carried out in PBS for 15 min.

Preparation of nuclei and cytosolic extracts

Nuclei from treated and untreated cells were prepared as described previously (Krude et al., 1997) with minor modifications. Briefly, cells were washed twice, incubated for 10 min in cold hypotonic buffer [20 mM Hepes pH 7.8, 5 mM potassium acetate, 0.5 mM MgCl2 and 1 mM DL-Dithiothreitol (DTT)] allowing their swelling. After buffer removal, cells were collected using a cell scraper and disrupted with twenty-five strokes in a Dounce homogenizer (Wheaton). Nuclei were pelleted at 3800 g for 2 min (Microfuge 18 centrifuge, Beckman Coulter), the supernatant was used for preparation of the cytosolic extract. Pelleted nuclei were washed twice in a 2× concentration of SuNaSp (20 mM Hepes pH 7.4, 150 mM NaCl, 0.5 M sucrose, 1 mM spermine, 0.3 mM spermidine and 6% BSA). After centrifugation at 3800 g for 2 min, nuclei were resuspended in an equal volume of 2×SuNaSp and counted in a Burker chamber. Cytosolic supernatant was centrifuged at 15,500 g for 20 min and the lipid portion was removed. The protein content, determined using a Bradford assay (Bradford, 1976), was 7–10 µg/µl. Nuclei and cytosolic extracts were then frozen by dropping them in liquid nitrogen.

Alternatively, a digitonin permeabilization protocol was used to obtain intact nuclei. In this case, treated and untreated cells were washed in cold PBS, trypsinized and collected in cold complete cell medium (DMEM supplemented with 10% FBS, 2mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin). After centrifugation at 180 g for 3 min at 4°C (Allegra 21R, Beckman Coulter), the pellet was washed with digitonin permeabilization buffer [50 mM KOH-Hepes pH 7.5, 50 mM K acetate, 5 mM Mg acetate, 2 mM DTT and 0.2 µl protease inhibitors (Sigma; cat. # P8340)], centrifuged at 88 g for 5 min at 4°C and resuspended in digitonin permeabilization buffer containing digitonin (100 µg/ml). Further increments of 5 µg/ml digitonin were added until the cells were permeabilized. The reaction was monitored by fluorescence microscopy, spotting on a coverslip 2 µl of cells mixed with 2 µl of fluorescein isothiocyanate–dextran (25 mg/ml). To block the permeabilization reaction, 10 ml of SuNaSp–BSA buffer [10 mM KOH-Hepes pH 7.4, 75 mM NaCl, 0.25 M sucrose, 0.5 mM spermine, 0.15 mM spermidine, 3% BSA, 2 mM DTT and 0.3 µl protease inhibitors (Sigma)] were added. After centrifugation, nuclei were resuspended in an equal volume of 2× SuNaSp and frozen by dropping them in liquid nitrogen.

Recombinant proteins

DDB2wt, DDB2PCNA− and PCNA His-tag recombinant proteins were expressed in DH5α E. coli and purified, as previously described (Cazzalini et al., 2014). The recombinant proteins DDB2wt (50, 100 and 150 ng), DDB2PCNA− (50, 100 and 150 ng), PCNA (400 ng) and DDB1 (150 ng, Abnova) were directly incubated for 10 min with the isolated nuclei before adding cytosolic extract. To make PCNA-depleted cytosol, the C-terminal p21 protein (p21C–GST), purified as previously published (Riva et al., 2004), was used. Pre-swollen glutathione-agarose beads (Sigma) were incubated for 1 h with p21C–GST protein. Then, the cytoplasmic extract was added and incubated for 1 h in a cold room. After centrifugation, the supernatant was collected and a second PCNA-depletion procedure was performed using new beads and p21C–GST complex. The samples for western blotting were collected after each depletion step. For permeabilization, nuclei were incubated in 0.1% Triton X-100 in SuNaSp–BSA buffer at 4°C in a rotator for 20 min then washed twice in SuNaSp–BSA buffer without Triton X-100.

In vitro DNA repair synthesis reaction

DNA repair synthesis reactions were performed in parallel by fluorescence microscopy and flow cytometry. Standard reaction conditions contained 10 µl of cytosolic extract (70–100 µg protein), 2 µl of nuclei (about 2×105) and 1.53 µl of reagent mix [yielding final concentrations of 40 mM K-HEPES (pH 7.8), 7 mM MgCl2, 3 mM ATP, 0.1 mM each of GTP, CTP and UTP, 0.1 mM each of dATP, dGTP, and dCTP, 0.25 μM biotin-16-dUTP (Roche), 0.5 mM DTT, 40 mM creatine phosphate and 5 μg phosphocreatine kinase (Calbiochem)]. The inhibition of DNA repair synthesis was evaluated by incubating nuclei in the standard reaction buffer in the presence of 1 mM aphidicolin (Sigma) and 20 µM Cytarabine (cytosine arabinoside; Sigma). Reactions were mixed on ice and started by transferring samples to 37°C. The standard incubation period was 2 h unless otherwise stated. For fluorescence microscopy, nuclei were permeabilized and fixed with 0.25% Triton X-100 (Sigma) and 4% formaldehyde for 10 min at room temperature, then stratified through a 30% sucrose gradient on coverslips previously treated with Poly-L-lysine solution (Sigma) by centrifugation at 180 g for 5 min (Allegra 21R, Beckman Coulter). For flow cytometry analysis, samples were prepared in the same experimental conditions, doubling the volumes and fixing them with 50% ethanol in physiological buffer (0.9% NaCl).

Immunofluorescence and flow cytometry

Coverslips were washed three times with blocking solution (BS; 0.02% Triton X-100, 0.04% SDS and 1% BSA in PBS), and incubated with streptavidin–Alexa Fluor 488 (1:350; S32354, lot 18585034, Invitrogen) for 30 min at 37°C, washed again with BS buffer and incubated with Hoechst 33258 dye (0.5 µg/ml) for 10 min at room temperature. For immunoreactions, coverslips were incubated with mouse monoclonal anti-BrdU (1:100; RPN202, clone BU-1, Amersham Pharmacia Biotech), according to the manufacturer's instructions, and anti-mouse Alexa Fluor 594 (1:200, RRID: AB_141607). Then, coverslips were mounted in Mowiol (Calbiochem) containing 0.25% 1,4-diazabicyclo-octane (Sigma-Aldrich) as antifading agent.

Samples for flow cytometry (about 5×105 nuclei) were pelleted at 180 g for 3 min (Allegra 21R, Beckman Coulter) and washed twice with BS buffer, then nuclei were incubated with streptavidin–Alexa Fluor 488 (1:200; S32354, lot 18585034, Invitrogen) for 30 min at room temperature, resuspending every 2 min. DNA was labelled with PBS containing 8.44 µg/ml propidium iodide (PI) and 0.05% Igepal CA 630 (Sigma) for 30 min at room temperature.

Western blotting

Nuclei were resuspended in 50 μl of PBS and sonicated. Both nuclei and cytosolic extracts of control and irradiated cells were quantified using the Bradford method (Bradford, 1976). For each sample, 30 μg of protein was mixed with a 3× SDS-loading buffer and electrophoresis was performed in denaturing and reducing conditions. DDB2 levels were detected using an anti-DDB2 antibody (1:1000, Santa Cruz; RRID: AB_2088827) and an HRP-conjugated anti-rabbit secondary antibody (Cat#A9169, lot#098M4861V, 1:10,000, Sigma). The chromatin-bound DDB2 was distinguished using an anti-histidine antibody (1:2000, Invitrogen), and PCNA levels were detected using an anti-PCNA antibody (1:1000, Dako, RRID:AB_2160651) and an HRP-conjugated anti-mouse secondary antibody (Cat#A9044, lot#029M4799V, 1:10,000, Sigma-Aldrich). Histone H3 was detected using an anti-H3 antibody (Cat#07-690, lot#2828613, 1:100,000, Millipore) and an HRP-conjugated anti-rabbit secondary antibody (Cat#A9169, lot#098M4861V, 1:10,000, Sigma-Aldrich). Phospho-histone H2AX was detected using anti-phospho-H2AX (Ser139; #07-164, 1:1000, Upstate) and an HRP-conjugated anti-rabbit secondary antibody (Cat#A9169, lot#098M4861V, 1:10,000, Sigma). H2AX was revealed using anti-H2A.X (Cat#SC-517336, clone 938CT5.1.1, 1:1000, Santa Cruz) and an HRP-conjugated anti-mouse secondary antibody (A9044, lot#029M4799V, 1:10,000, Sigma). To reveal protein levels, a chemiluminescent enhancer (Bio-Rad) was used. Densitometric analysis was performed using ImageJ software (NIH, MD).

Statistical analysis

All experiments were reproduced no less than three times. Results were expressed as mean±s.d. Statistical significance was calculated using a one-tailed Student’s t-test using Microsoft Excel.

We thank P. Vaghi (Centro Grandi Strumenti, Università di Pavia) for help in confocal microscopy analysis and S. Barbieri, M. Liotta and P. Tabarelli de Fatis for the technical assistance and support during the X-irradiation at IRCCS Maugeri (Pavia). We are very grateful to A. Lehmann (University of Sussex, Brighton, UK) for providing the XP14BR cell line and T. Nardo [Istituto di Genetica Molecolare (IGM) del CNR, Pavia, Italy] for her technical help with this cell line.

Author contributions

Conceptualization: O.C., L.A.S.; Investigation: I.G., E.B., M.S., P.P.; Writing - original draft: I.G.; Writing - review & editing: E.P., L.A.S.; Funding acquisition: L.A.S., E.P.

Funding

This research was supported by a grant from the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) to the Department of Molecular Medicine of the University of Pavia under the initiative ‘Dipartimenti di Eccellenza (2018–2022)’. This work was in part supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC; IG number 17041 to E.P.).

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Competing interests

The authors declare no competing or financial interests.

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