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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: DNA Repair (Amst). 2021 Sep 22;108:103230. doi: 10.1016/j.dnarep.2021.103230

Determination of DNA lesion bypass using a ChIP-based assay

Dayong Wu 1,2, Ananya Banerjee 1,2, Shurui Cai 1,2, Na Li 1,2, Chunhua Han 1,2, Xuetao Bai 1,2, Junran Zhang 1,2, Qi-En Wang 1,2,*
PMCID: PMC8616795  NIHMSID: NIHMS1742858  PMID: 34571449

Abstract

DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.

Keywords: Lesion bypass, translesion synthesis, TLS, chromatin immunoprecipitation, ChIP, cisplatin, cancer stem cell

1. Introduction

DNA replication is a highly regulated process that guarantees the faithful duplication of the genome DNA. Perturbation of DNA replication, including replication fork stalling induced by DNA damage, can lead to genome instability and cell death. Many chemotherapeutic drugs, such as cisplatin and carboplatin, kill cancer cells by inducing intrastrand crosslinks and interstrand crosslinks, which can serve as a barrier for DNA replication. Although platinum-DNA adducts can be repaired by the nucleotide excision repair pathway, some DNA lesions may not be efficiently repaired before they are encountered by replication forks, which can be stalled and replication stress occurs. However, these DNA lesions can be bypassed during DNA replication or post replication by specialized DNA polymerases (1), or through template switching (2). The translesion DNA synthesis (TLS) machinery, including replication-coupled bypass and post-replicative gap filling, is the major DNA damage tolerance mechanism that allows the cell to replicate over DNA lesions and ensures continuous DNA synthesis (3,4). As such, it is critical to the survival of cells following certain types of DNA damage, and contributes to chemotherapy resistance including cisplatin resistance (5–9).

TLS is mainly performed by a group of specialized DNA polymerases belonging to the Y and B families, including REV1, Polη, Polκ, Polι, and Polζ (10,11). These TLS polymerases resolve fork stalling at lesions in the template, thereby preventing the induction of double-strand breaks (DSBs). REV1 functions as a principal scaffolding protein, which recruits Polη, Polκ, or Polι through protein-protein interactions to insert a nucleotide opposite to the DNA lesion (12–15). REV1 also interacts with REV7 and POLD3 of the Polζ complex to facilitate extension beyond the insertion step (16,17). TLS is also facilitated by PCNA mono-ubiquitination (Ub-PCNA), which is regulated by Rad6-Rad18 E2-E3 ubiquitinating enzymes and USP1 deubiquitinating enzyme at sites of DNA damage (18–20). When replicative polymerases encounter DNA lesions, Rad6-Rad18 is recruited to the lesion and facilitates PCNA mono-ubiquitination. ub-PCNA can interact with the PCNA interacting protein (PIP) and ubiquitin-binding motif (UBM)/ubiquitin-binding zinc finger (UBZ) domains in Polη, Polκ, and Polι, promoting the recruitment of these TLS polymerases to the damage sites to bypass the lesion (21). Compared to replicative polymerases, TLS polymerases have low fidelity and can introduce inappropriate bases across from modified nucleotides, leading to the generation of mutations.

Currently, a variety of methods are used to evaluate lesion bypass activity. This activity can be indirectly assessed by determining the recruitment of TLS polymerases to replication forks, or by determining the amount of TLS polymerases and ub-PCNA in cells [Reviewed in (22)]. Lesion bypass can also be directly assessed in the cell-free system by determining DNA elongation using the primer extension assay (23–25). In addition, DNA replication progression in mammalian cells after DNA damage can be determined using the DNA fiber assay (26–28) and alkaline unwinding assay (ADU) (29) to indirectly reflect lesion bypass efficiency. DNA synthesis across DNA lesions can also be evaluated using a plasmid-based TLS assay, such as gapped plasmid TLS assay (30) or shuttle-vector-based strand-specific PCR-competitive replication and adduct bypass (SSPCR-CRAB) assay (31). However, all these currently used methods have limitations in direct evaluation of the lesion bypass activity across a specific DNA lesion in the context of mammalian cells. Here, we established a chromatin immunoprecipitation (ChIP) based lesion bypass method for directly analyzing the efficiency of replication bypass across cisplatin-induced intrastrand crosslinks in mammalian cells. By using this method, we provide direct evidence showing that ovarian cancer stem cells (CSCs) possess enhanced lesion bypass activity in bypassing cisplatin-induced DNA intrastrand crosslinks.

2. Materials and methods

2.1. Cell cultures and reagents

The human ovarian cancer cell line PEO1 was kindly provided by Dr. Rugang Zhang (The Wistar Institute), OVCAR3 was purchased from ATCC (Manassas, VA). All cell lines were authenticated by ATCC using DNA (short tandem repeat) profiling and tested for mycoplasma contamination on 1/22/2019. Cells were cultured in RPMI 1640 supplemented with 10% FBS (GIBCO/BRL), 100 μg/ml streptomycin and 100 units/ml penicillin. Cisplatin (Sigma) stock solution (4 mM) was prepared freshly with PBS and further diluted to the desired concentration with culture medium for cell treatment. JH-RE-06 was purchased from Aobiosu (Gloucester MA), and dissolved in DMSO.

2.2. Enrichment of CSCs by tumor sphere culture

PEO1 and OVCAR3 bulk cells were seeded in 60-mm ultra-low attachment dishes with serum-free 3D Tumorsphere Medium XF (C-28070, PromoCell). 0.5 ml of fresh medium was added to the culture every 2 days. Cell culture was centrifuged at 500 g for 5 min to harvest sphere cells when a single sphere contains more than 50 cells. The sphere cells were then separated by gently pipetting and reseeded into ultra-low attachment dishes for culture for at least 10 days before using as enriched CSCs.

2.3. Enrichment of CSCs by fluorescence-activated cell sorting (FACS)

Cellular aldehyde dehydrogenase (ALDH) activity was used to define CSCs in PEO1 cells. ALDH+ and ALDH− cells were sorted from PEO1 cells using the ALDEFLUOR kit (STEMCELL Technologies) on a BD FACS Aria III flow cytometer. A part of cells were treated with 50 mmol/L diethylaminobenzaldehyde (DEAB) to define the gate.

2.4. Small interference RNA (siRNA) and cell transfection

siRNA SMARTpools designed to target human POLH (L-006454) or REV1 (L-008234) and non-targeting control siRNA (5′-UUCUCCGAACGUGUCACGU-3′) were purchased from Horizon (Denver, CO, USA). shRNA targets REV1 (TRCN0000151738) and shRNA Control (SHC016) were purchased from Sigma-Aldrich (St. Louis, MO). For transfection, 5 ˣ 105 cells were seeded in a 60-mm dish 24 hr before transfection. When cells reached about 70% confluence, a total of 50 nM siRNA or 1 μg of shRNA was transfected into cells using the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

2.5. Western blot analysis

Whole cell lysates were prepared by boiling cell pellets for 10 min in a SDS lysis buffer (2% SDS, 10% glycerol, 10 mM DTT, 62 mM Tris–HCl pH 6.8, 10 μg/ml pepstatin and 1 μg/ml leupeptin). Proteins were quantitated, and 50 μg of protein were separated by SDS-PAGE gels, transferred to the nitrocellulose membrane, blocked with 5% FBS at room temperature for 1 h on a shaker. The membranes were incubated with the anti-Pol η antibody (1: 1000, A301–231A, Bethyl Laboratories), anti-REV1 antibody (1: 1000, PA5–46793, Thermo Fisher Scientific), anti-Ubiquityl-PCNA antibody (1:1000, #13439, Cell Signaling Technology), anti-Calnexin antibody (1:2000, ab92573, abcam), or anti-GAPDH antibody (1:2000, #97166, Cell Signaling Technology) overnight at 4°C. The membranes were washed with TBST, then incubated in an HRP-conjugated secondary antibody for 1 h at room temperature. After wash, the proteins were visualized by ECL Western blotting substrate (Thermo Fisher Scientific).

2.6. ChIP-lesion bypass assay

The ChIP assay was modified from the previously described (32). Briefly, 1 × 106 cells were treated with 20 μM cisplatin for 1 hr. Drug-containing medium was then removed, and cells were further cultured with 10 μM BrdU for 3 hrs in the absence of cisplatin. Cells were harvested and lysed in low-salt buffer (10 mM HEPES, pH 7.4, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 0.5% NP-40 and protease inhibitor) for 10 min at 4°C and chromatin was then sonicated or treated with micrococcal nuclease (MNase); For digestion with MNase, cell nuclei were resuspended in 100 μl MNase buffer containing MNase (Cat. No. M0247S, New England Biolabs) and incubated for 10 min at 37°C; DNA was extracted with phenol/chloroform and precipitated with ethanol. DNA was resuspended with TE buffer and 10– 30 μg of DNA was immunoprecipitated with 4 μg of anti-cisplatin modified DNA antibody (Rat anti-Pt-GG antibody, ab103261, abcam) at 4°C overnight. 30 μl of protein G-magnetic beads (Cat. No. 370024, Cell Signaling Technology) were added and further incubated for 1 hr on rotator. The beads were washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl) and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.1) and finally twice with TE buffer, then, resuspended in 100 μl buffer with 20 units of Exo VIII (Cat. No. M0299S, New England Biolabs) for 30 min. DNA bound to beads were extracted with 60 μl elution buffer (1% SDS, 0.1 M NaHCO3) in a Thermomixer and eluates were treated with proteinase K at 50°C overnight; DNA fragments were purified with the QIAquick PCR purification kit (Cat. No. 28104, Qiagen), and the DNA samples were boiled for 5 mins and immediately put on the ice; The samples were mixed with equal volumes of 2 M ammonium acetate and loaded on nitrocellulose membranes in the Hybri-Slot Vacuum Manifold (Cat. No. 1052MM, BRL); Membrane was dried and placed in a vacuum oven at 80 °C for 1 hr. Then the membrane was blotted with the rat anti-Pt-GG and mouse anti-BrdU antibody (Cat. No. MAB4072, Millipore Sigma), followed by incubation with the IRDye 800CW goat anti-rat-IgG (1:10,000) and IRDye 680RD goat anti-mouse-IgG (1:10,000) secondary antibodies (Li-Cor), respectively. The bands were visualized and the intensity of each band was determined using the Li-Cor Odyssey Imaging System. The ratio of BrdU incorporation to the cisplatin DNA adduct was calculated and used to represent the lesion bypass activity.

2.7. Statistical analysis

Results are presented as the mean ± SD. Statistical differences were determined by using two-sided T-test for independent samples. P < 0.05 was considered statistically significant.

3. Results

3.1. Development of the ChIP-based assay for assessing the activity of lesion bypass after cisplatin treatment

The major characteristic of lesion bypass is that specialized DNA polymerases synthesize DNA across the DNA damage site and allow replication to continue in the presence of such replication obstacles (33). Thus, successful lesion bypass results in the coexistence of DNA lesion and the newly synthesized DNA fragment opposite to this lesion (Fig. 1A). Based on this fact, we hypothesized that the amount of newly synthesized DNA coexisting with the DNA lesions can be used to assess the efficiency of lesion bypass in cells. To quantitate the amount of newly synthesized DNA coexisting with the DNA lesions, we need to label the newly synthesized DNA, purify the lesion-containing DNA fragments, and determine the quantity of labeled DNA in these fragments. ChIP is a procedure used to determine the binding or the colocalization of a given protein to a specific DNA sequence in cells. In this procedure, DNA-binding proteins are crosslinked to DNA, the chromatin is then sheared into small fragments. Antibodies specific to the DNA-binding protein are used to isolate the protein-DNA complex by precipitation, and the precipitated DNA is purified for further analysis. Borrowing from this idea, we proposed to precipitate DNA lesion-containing chromatin fragments using specific antibodies targeting DNA lesions, and determine the amount of newly synthesized DNA characterized by BrdU incorporation using immunoslot blotting (ISB) (Fig. 1B).

Fig. 1. Development of the ChIP-based lesion bypass assay with sonication.

Fig. 1.

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A. Schematic illustration of TLS across cisplatin-induced Pt-GG. When a replicative DNA polymerase encounters a blocking lesion such as Pt-GG, the polymerase dissociates, and a TLS polymerase binds to the DNA and incorporates a dNTP opposite to the lesion. After elongation, this TLS polymerase is replaced by the replicative polymerase and normal DNA replication resumes. B. Schematic illustration of the mechanism of lesion bypass analysis by the ChIP-based assay. BrdU is incorporated into newly synthesized strands that bypass Pt-GG DNA lesions during lesion bypass. Chromatin was broken to small fragments by sonication. Lesions containing DNA fragments are precipitated with the anti-Pt-GG antibody. Bypassed DNA fragments are purified and are subjected to immunoslot blotting (ISB) with the anti-BrdU antibody to determine the incorporated BrdU. C. The outline of cell treatment before the ChIP. PEO1 cells were treated with 20 μM cisplatin for 1 hr to generate DNA intrastrand crosslinks, or mock treated as a control. Cells were then incubated with 10 μM BrdU for 3 hrs in the absence of cisplatin, lysed and sonicated. D. DNA was isolated and DNA size was determined with agarose gel electrophoresis. E. The sonicated cell lysates were subjected to IP with either normal IgG or the anti-Pt-GG antibody. BrdU and Pt-GG in the immunoprecipitates were determine using ISB with their corresponding antibodies.

Given that cisplatin-induced intrastrand crosslink (e.g., Pt-GG) is an established lesion bypass substrate, we planned to precipitate Pt-GG-containing chromatin fragments using the anti-Pt-GG antibody, which is commercially available. Cells were treated with cisplatin for 1 hr to induce DNA damage, or mock treated as a control, and BrdU was added subsequently to label the newly synthesized DNA (Fig. 1C). The genomic DNA was sonicated to average 200 bp (Fig. 1D), and the immunoprecipitation was performed with the anti-Pt-GG antibody and protein G magnetic beads to isolate lesion-containing DNA fragments. The immunoprecipitated DNA was subjected to ISB for quantification of BrdU-containing DNA with the anti-BrdU antibody and the precipitated DNA lesion with the anti-Pt-GG antibody. It is clear that only the specific anti-Pt-GG antibody, and not the normal IgG, was able to enrich DNA fragments containing cisplatin-induced damage concomitant with incorporated BrdU in cells treated with cisplatin and labeled with BrdU; however, in the absence of cisplatin-induced DNA lesions, immunoprecipitation with the anti-Pt-GG antibody did not yield apparent Pt-GG nor BrdU signal, demonstrating the lesion-specific activity of the anti-Pt-GG antibody in this ChIP-based assay (Figure 1E). In summary, these data indicate that this procedure can be used to quantitate newly synthesized DNA coexisting with cisplatin-induced DNA lesions.

3.2. Optimization of the ChIP-based lesion bypass assay

It is possible that the already synthesized DNA before encountering DNA lesions may exist in the lesion-containing complex. These lesion bypass-independent BrdU-incorporated DNA may increase the non-specific background in determination of the lesion bypass-specific BrdU-incorporated DNA, and thus compromise the ability of this method to identify the minor difference between samples. To reduce the amount of lesion bypass-independent BrdU-incorporated DNA adjacent to the 5’-end of lesion bypass patch opposite to the cisplatin-induced crosslinks, we treated immunoprecipitates with truncated Exonuclease VIII (Exo VIII), which is a double-stranded DNA specific exonuclease and is able to catalyze the removal of nucleotides in the 5’ to 3’ direction until encountering the antibody bound to the cisplatin-induced intrastrand crosslinks (Fig. 2A) (34). Exo VIII treatment can significantly reduce the total BrdU-incorporated DNA precipitated with the anti-Pt-GG antibody, while the amount of Pt-GG was not affected (Fig. 2B). Given that DNA polymerase η (Polη) is required for TLS across cisplatin-induced intrastrand crosslinks, we attempted to compare lesion bypass efficiency between Polη downregulated and Polη intact cells in the presence or absence of Exo VIII treatment. Transfection of POLH siRNA significantly knocked down the expression of Polη in PEO1 cells (Fig. 2C), and resulted in a reduction in BrdU-incorporated DNA that are coexisting with Pt-GG both in Exo VIII treated and untreated cells (Fig. 2D, E). Exo VIII treatment significantly intensified the difference in the efficiency of lesion bypass between Polη downregulated and Polη intact cells (Fig. 2D–F), indicating that Exo VIII treatment can diminish the BrdU-incorporated DNA that is independent of lesion bypass, and thus enhance the potential of this assay to identify minor changes in lesion bypass efficiency.

Fig. 2. Enhance the sensitivity of the ChIP-based lesion bypass assay by removing lesion bypass-independent BrdU-incorporated DNA.

Fig. 2.

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A. Schematic illustration of Exo VIII digestion. B. The DNA immunoprecipitated with the anti-Pt-GG antibody, as described in Figure 2, was treated with Exo VIII or mock treated, then subjected to ISB for the analysis of BrdU and Pt-GG. C-F. Exo VIII treatment enhances the sensitivity of this assay to determine the reduction in lesion bypass activity. PEO1 cells were transfected with either control siRNA (siC) or POLH siRNA (siPOLH) for 48 hrs, treated with cisplatin and labeled with BrdU as described in Fig. 2. The knockdown of Polŋ expression was detected with immunoblotting (C); DNA isolated from immunoprecipitates were digested with Exo VIII or not digested, and the incorporated BrdU was determined using ISB. The immunoprecipitated Pt-GG was also detected to serve as a control (D). The intensity of BrdU and Pt-GG bands were measured, the BrdU band intensity relative to the corresponding Pt-GG was calculated to show the difference in BrdU incorporation between Exo VIII treated and mock treated cells (E). The ratio of the BrdU intensity relative to the corresponding Pt-GG in siPOLH cells and that in siC cells was calculated to represent the relative lesion bypass efficiency in siPOLH transfected cells after Exo VIII treatment and mock treatment (F). n = 3, Bar: SD, **: P < 0.01.

In a typical ChIP procedure, chromatin can be sheared with either sonication or MNase digestion. Sonication typically produces chromatin fragments between 200 and 500 bp, while MNase digestion normally produces chromatin fragments around 146 bp. Given that the size of chromatin fragments can affect the sensitivity of ChIP analysis (35), we sought to determine which DNA shearing method is more appropriate for the ChIP assay in assessing lesion bypass. Again, we compared lesion bypass efficiency between Polη downregulated and Polη intact cells when cells are sheared by sonication or MNase digestion. As expected, sonication of PEO1 cells yielded DNA fragments between 100–500 bp, while MNase digestion produced DNA fragments around 100–200 bp (Fig. 3A). When DNA was sheared by sonication, the ChIP-ISB analysis showed about 60% decrease in lesion bypass efficiency after siPOLH transfection. Meanwhile, MNase digestion led to a 75% decrease in lesion bypass efficiency after siPOLH transfection (Fig. 3B–D), indicating that differences in lesion bypass efficiency are more easily detected when DNA is fragmented by MNase digestion. In addition, we noticed that the lesion bypass efficiency in sonicated siPOLH cells in Fig. 3D is not same as that in Fig. 2F (+Exo VIII), indicating an inherent variability if sonication is used to fragment DNA, probably because compared to MNase digestion, sonication is not an efficient way to generate small DNA fragments of uniform length. Thus, we used MNase digestion instead of sonication in subsequent experiments assessing lesion bypass efficiency.

Fig. 3. Comparison of the effect of chromatin fragmentation on the sensitivity of ChIP-based lesion bypass assay.

Fig. 3.

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After treatment with cisplatin and labeled with BrdU as in Figure 2, PEO1 cell lysates were either sonicated or digested with MNase for 10 mins at 37°C. The size of DNA fragments isolated from two treatments was monitored using 1% agarose gel electrophoresis (A). Fragmented chromatin was subjected to the ChIP with the anti-Pt-GG antibody. After digestion with Exo VIII, the incorporated BrdU was determined using ISB. The immunoprecipitated Pt-GG was detected to serve as a control (B). The intensity of BrdU and Pt-GG bands were measured, the BrdU intensity relative to the corresponding Pt-GG (C) and lesion bypass efficiency in siPOLH transfected cells relative to the corresponding siC transfected cells (D) was calculated. n = 3, Bar: SD, **: P < 0.01.

3.3. Validation of the ChIP-based assay in assessing lesion bypass efficiency

Among the TLS polymerases, REV1 plays the most important role by serving as a scaffold that recruits and orchestrates other DNA polymerases involved in TLS of damaged DNA (15). Thus, we attempted to determine lesion bypass efficiency in REV1-proficient and REV1-deficient cells after cisplatin treatment using the ChIP-based assay. We transfected REV1 siRNA into OVCAR3 and PEO1 cells to knock down the expression of REV1 (Fig. 4A, D), and showed that downregulation of REV1 significantly inhibited replication across cisplatin-induced intrastrand crosslinks in both cell lines (Fig. 4B, C, E, F). We also validated this finding in OVCAR3 cells transfected with a REV1 shRNA that targets a different sequence of REV1 (Supplementary Fig. S1A–C). In a separate experiment, we inhibited TLS using a small molecule inhibitor, JH-RE-06, which disrupts the interaction between REV1 and Pol ζ (36). Its ability to inhibit replication across cisplatin-induced DNA damage has also been confirmed using the traditional gapped plasmid assay (36). We treated OVCAR3 and PEO1 cells with JH-RE-06 for 24 hr, and determined lesion bypass efficiency after cisplatin treatment using our ChIP-based assay. We were able to show about 50% and 80% inhibition of lesion bypass ability in OVCAR3 and PEO1 cells, respectively, by this small molecule (Fig. 4G–J). All these data indicate that our newly developed ChIP-based assay can be used to measure lesion bypass activity in cultured mammalian cells.

Fig. 4. Validation of the ChIP-based lesion bypass assay in ovarian cancer cells with TLS inhibition.

Fig. 4.

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OVCAR3 (A-C) and PEO1 (D-F) cells were transfected with either siC or siREV1 for 48 hrs. OVCAR3 (G, H) and PEO1 (I, J) cells were pretreated with 1.5 μM JH-RE-06 for 24 hrs. Cells were treated with cisplatin and BrdU as described in Fig. 2, MNase-digested cell lysates were subjected to the ChIP assay with the anti-Pt-GG antibody. (A, D) Knockdown of REV1 expression was detected using immunoblotting. (B, E, G, I) The immunoprecipitated DNA was digested with Exo VIII and subjected to ISB to detect the amount of incorporated BrdU and Pt-GG. (C, F, H, J) The intensity of BrdU and Pt-GG bands were measured and the relative lesion bypass activity was calculated. n = 3, Bar: SD, **: P < 0.01.

3.4. Ovarian cancer stem cells possess enhanced lesion bypass activity

Our previous study demonstrated that ovarian CSCs exhibit elevated Polη expression and increased ub-PCNA, suggesting that ovarian CSCs may possess enhanced TLS activity (37). To provide direct evidence to demonstrate the enhanced lesion bypass activity in ovarian CSCs, we enriched CSCs from ovarian cancer cell lines using various methods, analyzed and compared their ability to replicate across cisplatin-induced DNA damage to their corresponding bulk cells. CSCs can be identified and isolated based on their different characteristics, e.g., CSC-specific cell surface markers, side-population (SP) phenotypes by Hoechst 33342 exclusion, ability to grow as floating spheres in serum-free medium, and enhanced ALDH activity (38). Thus we enriched CSCs characterized by ALDH+ from PEO1 cells, as well as CSCs characterized by spheroid growth from OVCAR3 and PEO1 cells (Supplementary Fig. S2A). Cell cycle analysis showed that all these CSCs and their bulk cell counterparts have comparable percentages of cells in the S phase (Supplementary Fig. S2B). We also confirmed the enhanced expression of Polη and ub-PCNA in all CSCs compared to their counterparts (Supplementary Fig. S2C). The cells were treated with cisplatin followed by analyses of the replication across cisplatin-induced DNA damage. We showed that all CSCs are more efficient in replicating across cisplatin-induced intrastrand crosslinks compared to their counterparts (Fig. 5A–F). Furthermore, downregulation of REV1 was able to reduce the increased replication across DNA damage in CSCs (Fig. 5G, H). These data provide direct evidence to support our previous hypothesis that CSCs possess enhanced lesion bypass activity, probably through TLS.

Fig. 5. CSCs possess enhanced lesion bypass activity.

Fig. 5.

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A-F. ALDH+ and ALDH− cells isolated from PEO1 cells (A, B), as well as adherent and spheroid cultured OVCAR3 (C, D) and PEO1 (E, F) cells were treated with cisplatin, and labeled with BrdU as described in Fig. 2. Cell lysates were treated with MNase and subjected to the ChIP assay with the anti-Pt-GG antibody. The DNA isolated from immunoprecipitates were digested with Exo VIII, ISB was conducted to detect the amount of BrdU and Pt-GG (A, C, E). The relative lesion bypass activity was calculated by measuring the band intensity (B, D, F). G, H. Spheroid cultured OVCAR3 cells were transfected with shREV1 or shControl (shC) for 48 hrs. Cells were treated with cisplatin and BrdU, the ChIP assay was conducted to determine the lesion bypass activity as described above. n = 3, Bar: SD, **: P < 0.01.

To validate our finding that CSCs have enhanced lesion bypass, and compare the ChIP-based lesion bypass assay with a traditional method, we performed the DNA fiber assay, which is used to evaluate lesion bypass activity in live cells by analyzing DNA replication fork progression and fork stalling (Supplementary Fig. S3A). It has been reported that cells must be treated with a very high dose of cisplatin to achieve a detectable level of replication fork stalling for this assay (39). Indeed, although we found significant fork stalling after incubating cells with 300 μM of cisplatin for 1 h, we were unable to detect fork stalling for cisplatin doses less than or equal to 100 μM (Supplementary Fig. S3B, C). When we treated OVCAR3 CSCs enriched by sphere culture with 300 μM of cisplatin for 1 hr, no significant fork stalling was observed (Supplementary Fig. S3D, E), indicating that OVCAR3 CSCs must have a mechanism to resume cisplatin-induced fork stalling, probably through lesion bypass.

4. Discussion

DNA damage comes from different endogenous and exogenous sources such as alkylating agents, reactive oxygen species, and UV radiation. These damaged sites become obstacles for replicative polymerases during DNA replication. Although DNA damage can be repaired by a variety of DNA repair pathways, many lesions may not be repaired efficiently or in a timely manner due to multiple reasons such as lack of available DNA repair factors, and inaccessibililty of DNA lesions within the chromatin. Thus, a DNA damage tolerance mechanism such as lesion bypass plays a critical role in preventing replication perturbation-induced cell death. TLS not only bypasses bulky DNA lesions and prevents replication stalling, but is also engaged in filling single strand DNA gaps left behind by replicative polymerases (40). Thus, it should come as no surprise that enhanced TLS activity has been reported to contribute to chemotherapy resistance in various cancers (5–9).

TLS activity can be inferred indirectly by monitoring various TLS-related events inside the cell (22). For example, the ub-PCNA can increase following UV radiation or cisplatin treatment and plays a role in TLS across UV- and cisplatin-induced DNA intrastrand crosslinks (41,42). Thus, the amount of ub-PCNA and the interaction between chromatin-bound PCNA and TLS polymerases can be used to reflect TLS activity (18,43). The fiber assay (44) and the alkaline unwinding assay (45) are two methods used to assess the contribution of lesion bypass to DNA elongation during replication in mammalian cells. The gap-filling plasmid assay, which uses a lesion containing plasmid that cannot replicate in mammalian cells, mimics in vivo chromosome TLS, and specifically focuses on post-replicative TLS (46). However, all these methods have significant disadvantages. ub-PCNA is not required for all TLS (47,48), and unchanged ub-PCNA does not necessarily indicate low TLS activity. The fiber assay is widely used to determine DNA replication elongation and replication fork stalling, which may not be necessarily affected by lesion bypass activity. As a complement to the fiber assay, ADU mainly measures if DNA elongation is continuous from a pulse labelled area by using single strand DNA (ssDNA) ends at a replication fork as starting points for DNA unwinding in alkaline solution (49). This assay needs radioactive labeling, and cannot differentiate DNA repair-rescued replication forks and lesion bypass-rescued replication forks. In contrast, gapped plasmid method is used as a strong tool to evaluate the post-replicative TLS activity and also provides mutation information by sequencing the TLS region in the plasmids, which cannot be achieved by other methods. However, one disadvantage is that the procedure for gapped plasmid generation is complicated and some lesion-containing oligos like cisplatin DNA adduct are not commercially available. It also requires extremely high transfection efficiency of the gapped plasmids into cells, since the retrieved plasmids from transfected cells must be sufficient for bacteria transformation, and there must be enough colonies for TLS calculation.

The ChIP-based in vivo lesion bypass assay presented here is based on the idea that DNA fragments synthesized across a specific DNA lesion can be selectively enriched using antibodies that recognize the DNA adduct of interest, and the enriched sequences can be quantified to reflect the activity of lesion bypass in the genome in vivo. In this method, genome DNA is treated with cisplatin to generate Pt-GG DNA adducts, and subsequent DNA synthesis is labeled with BrdU during replication. The DNA is then sheared to small fragments and precipitated by lesion-specific antibody. The BrdU incorporation opposite to lesion sites is quantified to represent lesion bypass activity. The advantages of this method include being able to directly evaluate lesion bypass activity in the context of chromatin, and requiring relatively simple procedure that revolves around the immunoprecipitation assay, which is now a standard method in many laboratories. Unlike the fiber assay and ADU assay, this method can also identify stretches of nucleotides that are synthesized opposite a specific DNA lesion, e.g., cisplatin-induced Pt-GG intrastrand crosslink, therefore, enables us to directly assess the replication bypass over the specific DNA lesion. We have validated this method by inhibiting the TLS activity in cells with downregulation of REV1 and Polη, or with a TLS inhibitor. The reduced TLS capability to replicate across cisplatin-induced Pt-GG in these cells were successfully reflected by the amount of BrdU precipitated by the anti-Pt-GG antibody. We then tested this method in bulk ovarian cancer cells and their corresponding CSC subpopulations. We showed a higher lesion bypass activity in ovarian CSCs than that in bulk cancer cells, which is also supported by the traditional DNA fiber assay. This result further supports our previous finding that ovarian CSCs survive cisplatin treatment through enhanced lesion bypass, probably by elevated Polη expression and increased ub-PCNA in CSCs (37). Moreover, we observed a difference in lesion bypass between bulk cells and CSCs when they were treated with 20 μM of cisplatin using the ChIP-based lesion bypass assay, whereas 300 μM of cisplatin was needed before the difference could be detected by the DNA fiber assay. This suggests that our newly developed ChIP-lesion bypass assay is much more sensitive than the traditional fiber assay.

In this method, the size of DNA fragments is critical to the sensitivity of the assay. By using MNase digestion to reduce the fragment size to around 100–200 bp, and using the Exo VIII to remove lesion bypass-independent BrdU-incorporated DNA adjacent to the 5’-end of bypass patches opposite to the cisplatin-induced crosslinks, we successfully reduced the non-specific background, making it easier to detect minor changes in the lesion bypass activity.

Despite advantages of this method, limitations were nevertheless identified. The nascent DNA fragments around a specific DNA lesion detected in this method can result from direct extension by TLS of stalled forks, or from post-replicative TLS-mediated gap filling, or even from lesion bypass by template switching or repriming. Therefore, other methods may be needed to help evaluate the specific TLS activity. Additionally, this method can only be used to determine lesion bypass across DNA lesions for which a specific ChIP grade antibody is commercially available. Moreover, it cannot provide mutation information as conveniently as the gapped plasmid assay. Although sequencing of the precipitated DNA fragments might be conducted to provide mutation information, sequencing nascent BrdU-incorporated DNA that coexists with a strand containing a Pt-GG adduct would be a huge technical challenge.

Nevertheless, the ChIP-based assay developed here can be used to directly assess the ability of mammalian cells to replicate DNA across cisplatin-induced lesions that block typical replicative polymerases. Given that the bypass of these DNA replication barriers is predominantly carried out by the TLS, this method can be used to analyze the TLS activity after cisplatin treatment.

Supplementary Material

Supp.Materials

Highlights:

  • A ChIP-based assay was developed to determine the DNA lesion bypass

  • Cancer cells with inhibited TLS show reduced lesion bypass after cisplatin treatment

  • Ovarian CSCs exhibit high lesion bypass activity compared to bulk cancer cells

Cell cycle analysis

Cells were harvested and fixed with 70% ethanol, stained with staining solution (PBS containing 0.1% Triton X 100, 0.2 mg/ml RNase A, 20 μg/ml propidium iodine) at 37°C for 15 min. Cell cycle was analyzed using flow cytometry.

DNA fiber assay

Cells were pulse labeled with 50 μM of 5-iodo-2’-deoxyuridine (IdU, Sigma, #I7125) for 30 min, followed by 200 μM of 5-chloro-2’-deoxyuridine (CIdU, Sigma, #C6891) and cisplatin for 60 min. Cells were then harvested by trypsinization, and resuspended in PBS. 2.5 μl cell suspension was mixed with 7.5 μl lysis buffer (0.5% sodium dodecylsulfate, 200 mM Tris-HCl [pH 7.4], 50 mM EDTA), and dropped on the top of an uncoated glass slide. The slides were tilted to allow DNA to spread along the slide. Once dried, DNA spreads were fixed in methanol-acetic acid (3:1) solution for 10 min, and put in pre-chilled 70% ethanol at 4°C for at least 1 hr. DNA was then denatured with 2.5 mM HCl for 30 min at 37°C, blocked with 1% BAS in PBS for 30 min at room temperature (RT). IdU and CIdU were co-stained with mouse anti-BrdU antibody (BDS Biosciences, #347580, 1:200) and rat anti-CIdU antibody (Abcam Cat. No ab6326, 1:400), respectively at RT for 1 h. After washing with PBS with Tween (0.1% Tween), slides were further incubated with Alexa Fluor 488-conjugated goat anti-rat antibody (Invitrogen, A11006, 1:400) and Cy3-conjugated sheep anti-mouse antibody (Sigma-Aldrich, C2181, 1:400) for 1 h at RT. After washing, the slides were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and DNA fibers were imaged and measured using Revolve fluorescent microscope (ECHO, San Diego, CA). CIdU/IdU ratio was used to represent the progression of replication fork.

Acknowledgements

This work was supported by the National Institute of Health (R01CA211175 to QEW). The authors thank Dr. Rugang Zhang (The Wistar Institute) for kindly providing the PEO1 cell line. We also thank all members in the Wang lab for discussion.

Footnotes

Declaration of Competing Interest

None noted

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