
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39106163
10.1093/nar/gkae672
gkae672
AcademicSubjects/SCI00010
Genome Integrity, Repair and Replication
Redundant but essential functions of PARP1 and PARP2 in DNA ligase I-independent DNA replication
Bhandari Seema Khattri Cancer Research Facility, Departments of Internal Medicine and Molecular Genetics & Microbiology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 915 Camino de Salud, 1 University of New Mexico, Albuquerque, NM 87131, USA

Wiest Nathaniel Cancer Research Facility, Departments of Internal Medicine and Molecular Genetics & Microbiology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 915 Camino de Salud, 1 University of New Mexico, Albuquerque, NM 87131, USA

Sallmyr Annahita Cancer Research Facility, Departments of Internal Medicine and Molecular Genetics & Microbiology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 915 Camino de Salud, 1 University of New Mexico, Albuquerque, NM 87131, USA

Du Ruofei Cancer Research Facility, Departments of Internal Medicine and Molecular Genetics & Microbiology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 915 Camino de Salud, 1 University of New Mexico, Albuquerque, NM 87131, USA

https://orcid.org/0000-0002-2671-1711
Tomkinson Alan E Cancer Research Facility, Departments of Internal Medicine and Molecular Genetics & Microbiology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 915 Camino de Salud, 1 University of New Mexico, Albuquerque, NM 87131, USA

To whom correspondence should be addressed. Tel: +1 505 272 5404; Fax: +1 505 272 4039; Email: atomkinson@salud.unm.edu
23 9 2024
06 8 2024
06 8 2024
52 17 1034110354
23 7 2024
18 7 2024
09 2 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

While DNA ligase I (LigI) joins most Okazaki fragments, a backup pathway involving poly(ADP-ribose) synthesis, XRCC1 and DNA ligase IIIα (LigIIIα) functions along with the LigI-dependent pathway and is also capable of supporting DNA replication in the absence of LigI. Here we have addressed for the first time the roles of PARP1 and PARP2 in this pathway using isogenic null derivatives of mouse CH12F3 cells. While single and double null mutants of the parental cell line and single mutants of LIG1 null cells were viable, loss of both PARP1 and PARP2 was synthetically lethal with LigI deficiency. Thus, PARP1 and PARP2 have a redundant essential role in LigI-deficient cells. Interestingly, higher levels of PARP2 but not PARP1 associated with newly synthesized DNA in the LIG1 null cells and there was a much higher increase in PARP2 chromatin retention in LIG1 null cells incubated with the PARP inhibitor olaparib with this effect occurring independently of PARP1. Together our results suggest that PARP2 plays a major role in specific cell types that are more dependent upon the backup pathway to complete DNA replication and that PARP2 retention at unligated Okazaki fragments likely contributes to the side effects of current clinical PARP inhibitors.

Graphical Abstract

Graphical Abstract

National Institutes of Health 10.13039/100000002 R01 GM57479 R01 ES012512 R01 CA276837 P01 CA92584 University of New Mexico Cancer Center, an NCI-designated Comprehensive Cancer Center CA118100 NCI 10.13039/100000054 CA276837
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pmcIntroduction

In mammalian cells, the majority of Okazaki fragments are joined by DNA ligase I (LigI) (1,2), which is one of the three mammalian DNA ligases. While inactivation of the LIG1 gene is not compatible with mouse embryonic development (3,4), LIG1 null mouse B-cell lymphoma cells proliferate at a similar rate compared with wild-type cells indicating that there is an efficient LigI-independent backup pathway for lagging strand replication (5). A combination of genetic and cell biology studies have provided compelling evidence that this back-up pathway utilizes the LigIIIα-XRCC1 complex (6–10). In contrast to the mouse B-cell lymphoma cell lines (5), mouse LIG1 null primary embryonic fibroblasts grew more slowly than their wild-type counterparts, converted replication intermediates into high molecular weight DNA more slowly and had higher levels of genomic instability (4) indicating that, at least in some cell types, the LigIIIα-XRCC1 backup pathway does not fully substitute for the LigI-dependent pathway.

The best characterized roles of the LigIIIα-XRCC1 complex are in nuclear base excision and single-strand break repair (SSBR) (2,11,12). XRCC1 acts as a scaffold that binds to and coordinates the actions of multiple DNA repair enzymes to generate ligatable nicks for LigIIIα to complete the repair events in both of these repair pathways (13–16). In SSBR, the DNA damage-activated poly(ADP-ribose) polymerases (PARP)s, PARP1 and PARP2, catalyze poly ADP-ribosylation of themselves and adjacent proteins using NAD+ as a substrate after binding to DNA breaks (17–20). The LigIIIα/XRCC1 complex along with associated repair enzymes is recruited to the DNA damage site by the binding of XRCC1 to the poly(ADP-ribose) (PAR) generated by PARP1 and, to a lesser extent, PARP2 in the vicinity of the DNA break (21–24). While this recruitment is not essential for SSBR, the absence of PARP1, which is more abundant than PARP2 and responsible for the majority of poly(ADP-ribose) synthesized in response to DNA damage, results in a significant reduction in the rate of SSBR (17,23–25).

Interestingly, there are increased levels of DNA ligase IIIα and XRCC1 as well as poly(ADP-ribose) (PAR) at replication foci in human cells with reduced LigI activity suggesting that SSBs between unligated Okazaki fragments stimulate PAR synthesis to recruit the LigIIIα/XRCC1 complex in a manner similar to its recruitment to SSBs generated by DNA damaging agents (7,8,26). Using a modified version of iPOND (9,27), we examined the association of proteins with and release from newly synthesized DNA in parental and LIG1 null mouse B cells (9). Surprisingly, there were remarkably few changes in the replication machinery in the absence of LigI. No differences were detected for the PCNA sliding clamp, the PCNA clamp loader, the PCNA clamp unloader, the Pol ϵ catalytic subunit, DNA maintenance methylation proteins and core histones, although there were elevated levels of the Pol δ catalytic subunit (9). As expected, there were elevated levels of DNA ligase IIIα and XRCC1 associated with newly synthesized DNA in the LIG1 null cells. Notably, the release of DNA ligase IIIα and XRCC1 from the newly synthesized DNA occurred with similar kinetics to PCNA and other replication proteins (9), suggesting that the DNA ligase IIIα-XRCC1 complex is acting and moving with the replication machinery rather than joining single breaks behind the replication machinery resulting from the failure to join Okazaki fragments.

LigI-deficient human cells are hypersensitive to inhibition of PAR synthesis (28,29). Furthermore, studies with an inhibitor of poly(ADP-ribose) glycohydrolase (PARG), the main cellular enzyme responsible for degrading PAR, showed that PAR synthesis occurs at replication sites even in cells with wild-type LigI function (8). Thus, it appears that there is a PAR and LigIIIα/XRCC1-dependent pathway for joining Okazaki fragments that is active even during unperturbed DNA replication (8,26). Neither PARP1 nor PARP2 is required for mouse embryonic development but deletion of both genes results in embryonic lethality at day 8.0 indicating that PARP1 and PARP2 have essential but functionally redundant roles in embryogenesis (30). Since human retinal epithelial cells lacking both PARP1 and PARP2 are viable, DNA replication can occur in the absence of both PARP1 and PARP2 at least in some cell types (8). This is consistent with the relatively mild side-effects of PARP inhibitors, such as olaparib, that inhibit both PARP1 and PARP2 (31).

The PARP inhibitors were initially developed to selectively target homology-dependent repair (HDR)-deficient tumors in women with inherited predisposition to breast and ovarian cancer by generating replication-dependent one-ended DSBs as a consequence of inhibiting SSBR (32,33). It has, however, become evident that these inhibitors have pleiotropic effects. Specifically, it has been shown that PARP inhibitors also increase replication fork speed, generate replication-dependent single strand gaps, increase the retention of PARP1 and PARP2 on chromatin and cause transcription-replication conflicts (34–38). While the cytotoxicity of PARP inhibitors correlates with their ability to retain PARP on chromatin (34), the exact molecular mechanism of PARP retention, also referred to as PARP trapping, as well as the nature of endogenous DNA substrates where PARP retention occurs remains poorly understood (39–42). Notably, recent studies have suggested that unligated Okazaki fragments are the source of cytotoxic PARP1 trapping (26) and that replication-dependent single-strand gaps are generated as a result of PARP trapping (36). Here, we have used a combination of genetic and chemical inhibition to examine the interplay between LigI, PARP1 and PARP2. Together our results suggest that, while PAR synthesis by either PARP1 or PARP2 is essential in LIG1 null cells, PARP2 plays a major role in the LigIIIα/XRCC1-dependent backup pathway for joining Okazaki fragments in specific cell types that are more dependent upon the backup pathway to complete DNA replication and that PARP2 retention at unligated Okazaki fragments likely contributes to the side effects of clinically relevant PARP inhibitors.

Materials and methods

Cell culture

Wild-type and LIG1 null CH12F3 cell lines (5) and derivatives of the LIG1 null cells stably expressing either wild-type LigI or the non-methylatable TARK mutant version of LigI (43) have been described previously. All the cell lines were grown in RPMI Medium 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and freshly added 55 μM β-mercaptoethanol at 37°C in a humidified atmosphere with 5% (v/v) CO2. Where indicated, cells were incubated with the PARG inhibitor, PDD 00017273 (Tocris, cat. no. 5952) and the PARP inhibitors, olaparib (ApexBio, cat. no. A4154), AG-14361 (Selleck chemicals, cat. no. S2178) and UPF 1069 (Selleck chemicals, cat. no. S8038).

Antibodies

The following commercial antibodies were used at the indicated dilution for immunocytochemistry or immunoblotting; rabbit anti-Ligase III (Cell Signaling, cat. no. 103172S, 1:1000); rabbit anti-PARP1(Cell Signaling, cat. no. 9532S, 1:2000); mouse anti-PARP2 (Enzo Life Sciences, cat. no. ALX-804-639-L001, 1:50); mouse anti-XRCC1 (Santa Cruz, cat. no. sc56254, 1:200); mouse anti-PCNA (Santa Cruz, cat. no. sc56, 1:1000); anti-pan-ADP-ribose binding reagent (Millipore sigma, cat. no. MABE1016, 1:2000); rabbit anti-pADPr (Cell Signaling, cat. no. 83732S, 1:500); rabbit anti-beta-tubulin (Cell Signaling, cat. no. 2148S, 1:1000); rabbit mAb anti-H3, HRP conjugate (Cell Signaling, cat. no. 12648S, 1:5000); Alexa Fluor 488 goat anti-mouse IgG2a (Invitrogen, cat. no. A21131); Alexa Fluor 568 goat anti-rabbit IgG (H + L) (Invitrogen, cat. no. A11011). The polyclonal rabbit anti-LigI, which was raised against purified human LigI, has been described previously (44), was used at a 1:5000 dilution.

Immunocytochemistry

After incubation with either 10 μM of the PARG inhibitor PDD 00017273 or DMSO for 20 min, a monolayer of cells was deposited on slides using cytospin (800 × g for 3 min) prior to immunostaining as previously described (8). Briefly, cells were pre-extracted on ice for 2 min using 0.2% triton and then fixed with 4% formaldehyde/PBS for 10 min at room temperature. After washing with 1× PBS (2 × 5 min), cells were permeabilized by treatment with ice-cold methanol/acetone solution (1:1) for 5 min, washed with 1× PBS (2 × 5 min) and then blocked with 5% BSA for 45 min at room temperature. Slides were rinsed with 1× PBS (2 × 5 min) and then incubated with first primary antibody, anti-PCNA (Santa Cruz, sc-56, 1:50), for 60 min at room temperature. After rinsing with 1× PBS (4 × 5 min), slides were incubated with the second primary antibody, anti-pADPr (Cell Signaling, cat. no. 83732S, 1:500), for 60 min at room temperature. Slides were then rinsed with 1× PBS (4 × 5 min) followed by incubation with first secondary antibody, Alexa Fluor 488 goat anti-mouse IgG2a (Invitrogen, cat. no. A21131, 1:500), for 60 min at room temperature. After rinsing with 1× PBS (3 × 5 min), slides were incubated with the second secondary antibody, Alexa Fluor 568 goat anti-Rabbit IgG (H + L) (Invitrogen, cat. no. A11011, 1:1000), for 45 min at room temperature. Slides were then rinsed sequentially with 1× PBS (3 × 5 min) and distilled water (1 × 2 min) before the coverslip was mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, cat. no. P36935) and allowed to dry. Cells were imaged using a Leica TCS-SP8 (Leica Microsystems; Wetzlar, Germany) controlled by LASX software version 3.5.7 followed by computational image restoration (deconvolution) with Huygens Essential version 20.10 (Scientific Volume Imaging; Hilversum, Netherland) utilizing a constrained maximum likelihood estimation algorithm. LAS X software was used to convert all deconvolved images to maximum intensity projections (MIPs) which were then analyzed using Slidebook 6 version 6.0.6. For the double-staining combination, approximately 30 cells were imaged per cell line in three independent experiments.

Whole cell lysate (WCL) preparation and immunoblotting

Clarified whole cells lysates were prepared using RIPA buffer supplemented with 1× protease inhibitor cocktail (Sigma-Aldrich, cat. no. P8340) and 1 mM PMSF as described (9). Protein concentrations of the lysates were determined using the assay described by Bradford (45) (Bio-Rad, cat. no. 500-0006). Proteins were separated by SDS polyacrylamide gel-electrophoresis (PAGE) (46) and transferred onto PVDF membranes. After blocking with 5% milk in TBST for 1h at room temperature, membranes were incubated with specific primary antibodies. The membranes were washed (4 × 5 min) with 1× TBST, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Following 4 × 5 min washes with 1× TBST, peroxidase activity was detected using ECL reagent (Cytiva, cat. no. RPN2106).

aniPOND-SILAC MS

Identification of proteins by mass spectrometry following aniPOND was performed as described previously (9,47). Briefly, CH12F3 wild-type and LIG1 null cells grown either in light or heavy SILAC media were pulse-labeled with EdU and then chased with thymidine (9). After lysis and prior to the click reaction, samples from light and heavy labeled cells were mixed 1:1 and then separated by SDS-PAGE prior to staining with Coomassie blue (9). Gel regions above and below the streptavidin band were excised and then processed as described (9,48). Tryptic peptides were separated by MudPIT using an 8-step salt gradient and then identified by mass spectrometry (MS) in the Vanderbilt Mass Spectrometry Research Center Proteomics Core (48). For peptide and protein identification, data were analyzed using the Maxquant software package, version 1.3.0.5 with MS/MS spectra searched against the UniprotKB protein database. Peptide intensities for specific proteins were normalized by dividing by the intensity value for H4 histone peptides. The ratio of the normalized values for a protein were calculated by the dividing normalized values from the heavy isotope-labeled extract from the LIG1 null cells by the normalized values from the light isotope-labeled extract from the wild-type parental cells (Run 1). Similar experiments were carried out in which the heavy and light isotope media were switched (Run 2). The calculations and graphics were performed by programming in R 4.0.1 using the free platform RStudio 2022.02.03.

CRISPR-Cas9 gene editing

For gene editing, CH12F3 cells were transfected with Cas9 and the following CRISPR-Cas9 guide RNAs (Integrated DNA Technologies);

PARP1: 5′-CGGAGTACGCCAAGTCCAAC-3′

PARP2: 5′-GATCTGTAACGTGCAGACCA-3′

LIG1: 5′-GACGCTTTGGGAATCCTGAT-3′

using Invitrogen neon transfection system (Thermofisher scientific, cat. no. MPK10096) and incubated at 37 °C in 5% CO2. After 24 h, ATTO550 positive cells were sorted into 96-well plates either at 2000 cells/well for CyQUANT assays or at ∼1 cell/well for clone selection. The remaining ATTO550 positive cells were collected in 6-well plates, incubated at 37 °C in 5% CO2 for 72 h and then lysed for immunoblotting as described above. For single cell clones, the plates were incubated until sufficient cell density was achieved for analysis by immunoblotting.

CyQUANT assays

Cellular proliferation was measured using the CyQuant NF reagent (CyQUANT NF cell proliferation assay kit, Thermofisher scientific, cat. no. C35006). Cells were added at 2000 cells/well to 96-well plates and then incubated with varying concentrations of the PARP inhibitors, Olaparib, AG14361 or UPF1069 (or DMSO alone as a control) at 37°C in 5% CO2 for 72 h. After centrifugation at 400 × g for 10 min, the medium was removed and the CyQuant NF reagent was added to the wells and incubated according to manufacturer's instructions. Fluorescence intensity was measured using excitation at 485 ± 10 nm and fluorescence detection at 530 ± 15 nm in a microplate reader. Cell number is expressed as a percentage of the value obtained with DMSO-treated cells.

Colony forming assays

Colony forming assays were performed as described previously (49). Cells (300 cells/well) were suspended in 500 μl of cell culture medium (RPMI Medium 1640, 10% FBS, 1% penicillin/streptomycin and 55 μM β-mercaptoethanol) containing varying concentrations of the PARP inhibitors, olaparib, AG14361 or UPF1069 (or DMSO alone as a control). Methylcellulose-based media (3.5 ml of a 1:3 dilution of the methylcellulose stock solution (R&D Systems, cat. no. HSC0010 with the cell culture medium) (Methylcellulose stock solution,) was then added to the cell suspension and briefly vortexed. After sitting for 30 minutes, the cell suspensions were plated in 6-well plates and incubated for 10–14 days at 37 °C in 5% CO2. The colonies were then stained by adding 1 ml of iodonitrotetrazolium (1 mg/ml) (Sigma-Aldrich, cat. no. I10406) and incubating overnight. Colonies were counted manually.

Subcellular fractionation

Subcellular fractionation was performed as previously described with some modifications (7). Cells (∼1.5 × 107) were washed with 1× PBS and then lysed by incubating with ice cold buffer A (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM Pipes pH 6.8, 1 mM EGTA, 0.5% Triton X-100) supplemented with protease inhibitor cocktail 1 (Sigma-Aldrich, cat. no. P8340), 1 mM DTT for 5 minutes on ice. Following centrifugation, the supernatant was collected as the soluble fraction. After washing twice with ice cold 1× PBS, the pellet was resuspended in buffer B (50 mM Tris–HCl, pH 7.5, 20 mM NaCl, 1 mM MgCl2, 0.1% SDS) supplemented with 25U of the DNase Benzonase (EMD Millipore, cat. no. 70746-4) and protease inhibitor cocktail 1 (Sigma-Aldrich, cat. no. P8340) and incubated at room temperature for 15 min with occasional tapping prior to sonication on ice (2 rounds of 10 sec on and 30 sec off at 10W output) to shear the DNA and generate the chromatin fraction. Soluble and chromatin fractions were mixed with 2× Laemmli buffer (with 5% v/v β-mercaptoethanol) prior to boiling at 95°C for 5 min and separation by SDS-PAGE (46).

Results

LIG1 null cells have elevated levels of spontaneous DNA damage

Given the predominant role of LigI in joining Okazaki fragments (1,2), the viability of LIG1 null mouse CH12F3 cells and the absence of a proliferative defect was surprising (5). Since both human lig1 mutant fibroblasts and mouse LIG1 null embryonic fibroblasts have elevated levels of spontaneous DNA damage (4,50), we considered the possibility that, while LigIIIα-dependent DNA replication enabled the LIG1 null mouse CH12F3 cells to proliferate at the same rate as the wild-type cells, it may also result in increased steady state levels of DNA damage. Therefore, we examined the steady state levels of poly(ADP-ribose) (PAR), an indicator of SSBs. To detect SSBs generated during lagging strand DNA synthesis, PAR signals were quantified in S phase cells identified by presence of chromatin-bound PCNA foci (8). PAR synthesis was only detectable in wild-type CH12F3 cells following incubation with a PARG inhibitor whereas PAR synthesis was detectable in untreated CH12F3 LIG1 null cells and was present at markedly higher levels than in wild-type cells following incubation with a PARG inhibitor (Figure 1A and B). Thus, it appears that there are elevated steady state levels of SSBs in cells that are dependent upon LigIIIα for Okazaki fragment joining and that, as was observed in human retinal pigment epithelial cells (8), a fraction of Okazaki fragments are joined by the LigIIIα-dependent pathway in cells with fully functional LigI. Since the presence of genomic SSBs can lead to the formation of DSBs, we also measured the steady state levels of γH2AX, an indicator of DSBs, as well as PAR by immunoblotting. As expected, the CH12F3 LIG1 null cells had higher levels of both γH2AX and PAR than the parental wild-type cells (Figure 1C). Furthermore, expression of either wild-type LigI or a mutant version of LigI that is defective in recruiting the DNA methylation machinery to replication foci (43) reduced the levels of the DNA damage biomarkers to those observed in wild-type CH12F3 cells (Figure 1B and C).

Figure 1. LIG1 null cells have elevated levels of spontaneous DNA damage. (A) Representative confocal images of DAPI (blue), PCNA (green) and ADP-ribose (red) immunostaining in CH12F3 wild-type cells (WT) and LIG1 null derivative (LIG1−/−) following 20 min incubation with DMSO (vehicle) or the PARG inhibitor (PARGi) PDD 00017273 (10 μM). (B) Quantification of ADP-ribose levels in CH12F3-WT, LIG1-/- and LIG1 null cells expressing Flag-tagged wild-type LigI (KO + WT) cells incubated with either DMSO or PARG inhibitor. Data are the mean (±) SEM of three independent experiments. (C) Immunoblots of whole cell extracts (50 μg) from CH12F3 wild-type (WT), LIG1−/−, and LIG1−/– cells expressing either Flag-tagged wild-type LigI (KO + WT) or the TARK mutant version (KO + K126R) with the indicated antibodies.

Elevated levels of PARP2 but not PARP1 associated with newly synthesized DNA in LIG1 null cells

Previously, we utilized a modified version of iPOND, aniPOND, coupled with SILAC-MS to show that, as expected, elevated levels of LigIIIα and XRCC1 were associated with newly synthesized DNA in CH12F3 LIG1 null cells compared with the parental wild-type cells, whereas there were no detectable changes in the majority of replication proteins (9). Since replicating LIG1 null cells had higher levels of PAR (Figure 1), we examined the association of PARP1 and PARP2 with newly synthesized DNA (Figure 2). The SILAC labeling media was switched to confirm that changes observed were not due to the difference in the media (Figure 2, compare upper and lower panels). Following pulse labeling with EdU, similar amounts of PARP1 were associated with the EdU-labeled, newly synthesized DNA in the wild-type and LIG1 null cells (Figure 2A). These levels declined to a similar extent in both cell lines during the thymidine chase period indicating that PARP1 is associated with the replication machinery (Figure 2A). Interestingly, there was a more than 3-fold higher amount of PARP2 associated with the EdU-labeled, newly synthesized DNA in the LIG1 null cells compared with the wild-type cells (Figure 2B). As with PARP1 (Figure 2A), the levels of PARP2 associated with the EdU-labeled DNA in the LIG1 null cells declined during the thymidine chase period indicating that PARP2 is also associated with the replication machinery (Figure 2B). While it has been shown previously that ATM is activated in LigI-deficient cells (50) and there are increased steady state levels of γH2AX in LigI-deficient (50) and LIG1 null cells (Figure 1), we did not reproducibly detect higher levels of other DNA damage response proteins associated with the newly synthesized DNA in the LIG1 null cells.

Figure 2. Elevated level of PARP2 with replicating DNA in LIG1 null cells. The relative levels of: (A) PARP1 and (B) PARP2 associated with EdU-labeled DNA in CH12F3 wild-type (WT) cells and a LIG1 null derivative (LigI) were determined by aniPOND coupled with mass spectrometry as described in Materials and Methods. In the upper panels, the wild-type cells were grown in the heavy medium (WT: H, blue line) whereas the LIG1 null cells (LigI: L, red line) were grown in the light medium. In the lower panels, the wild-type cells were grown in the light medium (WT: L, blue line) whereas the LIG1 null cells (LigI: H, red line) were grown in the heavy medium.

Loss of both PARP1 and PARP2 is synthetically lethal in LIG1 null cells

The elevated levels of PARP2 associated with the newly synthesized DNA in LIG1 null cells suggests that PARP2 has a specific role in LigI-independent DNA replication. To examine this possibility, we examined the effect of reducing PARP2 activity in LIG1 null mouse cells. CRISPR-Cas9 targeting of PARP2 had only a minor effect on the proliferation of the LIG1 null cells (Supplementary Figure S1A and C) and the parental wild-type cells (Supplementary Figure S1A and B). Similarly, targeting of PARP1 also did not impact the proliferation of cultures of either LIG1 null cells or the parental wild-type cells (Supplementary Figure S1A-C). However, when we combined the guide RNAs for both PARP1 and PARP2, the growth of the LIG1 null cells (Supplementary Figure S1A and C) but not the parental wild-type cells (Supplementary Figure S1A and B) was notably reduced. While neither PARP1 nor PARP2 were detectable in the doubly-targeted proliferating culture of wild-type cells (Supplementary Figure S1A and B), PARP1 was expressed at low levels in the doubly-targeted, slow growing culture of LIG1 null cells (Supplementary Figure S1A and C). This prompted us to isolate individual clones of CH12F3 wild-type and LIG1 null cells lacking either PARP1 or PARP2 (Figure 3A). As expected, the clones with PARP1 knocked out had much lower levels of protein ADP-ribosylation (Figure 3A and C). Using the PARP single mutants, we attempted to knock out the other PARP gene. Targeting the other PARP gene did not impact the proliferation of the wild-type CH12F3 cells (Supplementary Figure S2A and B). In contrast, targeting the other PARP gene in the LIG1 null cells dramatically reduced proliferation (Figure 3B and C). While we were able to isolate several double knockout clones in the wild-type background (Supplementary Figure S3, upper panels), all surviving LIG1 null clones had residual PARP1 or PARP2 expression (Supplementary Figure S3, lower panels). These results strongly suggest that, in the absence of LigI, either PARP1 or PARP2 is required for cell viability. To further support this conclusion, we carried out reciprocal experiments, in which LIG1 was targeted in wild-type CH12F3 cells and the PARP single and double knockout derivatives (Figure 4A). The proliferation and survival of the double knockout cells but not the parental or single mutants was significantly reduced by LIG1 targeting (Figure 4B-D and Supplementary Figure S4). Thus, we conclude that PARP1 and PARP2 have functionally redundant roles in the LigIIIα-XRCC1 dependent backup pathway for joining Okazaki fragments that is essential in the absence of LigI.

Figure 3. Loss of both PARP1 and PARP2 is synthetically lethal in LIG1 null cells. (A) PARP1 (PARP1−/−) and PARP2 (PARP2−/−) knockout clones were generated in CH12F3 wild-type cells (WT) and a LIG1 null derivative (LIG1−/−) using CRISPR-Cas9 technology and verified for loss of targeted protein by immunoblotting. (B) LIG1−/− PARP1−/-and LIG1−/− PARP2−/-cells were targeted with sgRNA specific for PARP2 (PARP2 sgRNA) and PARP1 (PARP1 sgRNA), respectively and loss of the targeted protein verified by immunoblotting. (C) Effect of loss of PARP proteins on the proliferation of the LIG1 null (LIG1−/−) cells was measured by the CyQUANT assay as described in Materials and methods and shown graphically. Data are the mean (±) SEM of three independent experiments. Unpaired two-tailed Student's t-test. ‘*’ P-values < 0.05, ‘**’ P-values < 0.01, ‘***’P-values < 0.001.

Figure 4. Loss of Lig1 is not compatible with survival of PARP1 PARP2 double knockout cells. (A) CH12F3 wild-type cells (WT) and derivatives lacking PARP1 (PARP1−/-), PARP2 (PARP2−/−) or both PARP1 and PARP2 (PARP1/PARP2−/−) were targeted with a Lig1 sgRNA. Expression of LigI was determined by immunoblotting. (B). Effect of loss or reduced expression of Lig1 protein on the proliferation of CH12F3 wild-type (WT) cells and the PARP knockout derivatives was measured by the CyQUANT assay as described in Materials and methods. (C) Effect of loss or reduced expression of Lig1 protein on colony formation by CH12F3 wild-type(WT) cells and the PARP knockout derivatives. (D) Results of the colony forming assays are shown graphically. Data are the mean (±) SEM of three independent experiments. Unpaired two-tailed Student's t-test. ‘*’ P-values < 0.05, ‘**’ P-values < 0.01.

LIG1 null cells are hypersensitive to PARP inhibitors

In accord with the increased sensitivity of the human LigI-deficient 46BR.1G1 cell line to the PARP inhibitor 3-aminobenzamide (28,51), the LIG1 null mouse CH12F3 cells exhibited reduced proliferation and cell survival compared to the wild-type parental cells and a complemented derivative of the LIG1 null cells when incubated with the PARP1/2 inhibitor olaparib (31) (Figure 5A-C). Similar results were obtained with an inhibitor, AG14361 (Figure 5D–F) that is reported to be selective for PARP1 (32,52), whereas an inhibitor, UPF 1069, that is reported to be selective for PARP2 (53) did not significantly reduce either the growth or survival of any of the cell lines (Supplementary Figure S5).

Figure 5. LIG1 null cells are hypersensitive to PARP inhibitors. CH12F3 wild-type cells (WT) and CH12F3 LIG1 null cells (Lig1 null) as well as derivatives of the LIG1 null cells that stably express Flag-tagged wild-type LigI (KO + WT) cells were incubated with either the PARP1/2 inhibitor, olaparib or the PARP1 selective inhibitor AG14361 as indicated. (A and D) Effect of the PARP inhibitors on cell proliferation was measured by the CyQUANT assay. (B and E) Effect of the PARP inhibitors was also measured by colony formation. (C and F) Results of the colony formation assay are shown graphically. Data are the mean (±) SEM of three independent experiments. Unpaired two-tailed Student's t-test. ‘*’ P-values < 0.05, ‘**’ P-values < 0.01, ‘***’P-values < 0.001.

PARP inhibitors enhance the retention of PARP2 on chromatin in LIG1 null cells

Since the LigIIIα-XRCC1 complex is recruited to sites of DNA damage in a PAR-dependent manner (21–24), we examined the effects of PARP inhibition on the levels of chromatin-bound XRCC1 and LigIIIα in wild-type and LIG1 null cells. In accord with our previous study (9), chromatin-bound levels of XRCC1 and LigIIIα were significantly higher in untreated LIG1 null cells compared with wild-type cells (Figure 6A–C). As expected, treatment with olaparib reduced the levels of PAR and chromatin-bound XRCC1 in both WT and LIG1 null cells (Figure 6A and B). We did not, however, observe a significant reduction in chromatin-bound LigIIIα in either wild-type or LIG1 null cells (Figure 6C). Since XRCC1 functions as a binding partner for many DNA repair enzymes and it has an S phase function that is independent of LigIIIα (13,14,54), it is likely that there is pool of XRCC1 that is not complexed with LigIIIα and that this pool may be dependent upon parylation for chromatin recruitment whereas LigIIIα-XRCC1 may be retained on chromatin via protein-protein interactions with either PARP1 or PARP2 (25,55,56).

Figure 6. Effect of Olaparib on chromatin binding of PARP1 and PARP2. (A) Immunoblots of soluble and chromatin fractions obtained from CH12F3 wild-type cells (WT) and a LIG1 null derivative (LIG1-/-) incubated for 4 h with either DMSO (vehicle control) or olaparib (10 μM), with the indicated antibodies. Quantification of the steady state levels of (B) XRCC1; (C) LigIIIα (D) PARP1 and (E) PARP2 in the chromatin fraction of WT and LIG1−/− cells normalized to corresponding H3 levels. Data are the mean (±) SEM of three or four independent experiments. Unpaired two-tailed Student's t-test. ‘*’ P-values < 0.05, ‘**’ P-values < 0.01, ‘***’P-values < 0.001.

While treatment with olaparib did not result in a significant increase in the retention or trapping of either PARP1 or PARP2 on the chromatin of wild-type cells (Figure 6A, D and E), PARP1 and, to an even greater extent, PARP2 were retained at significantly higher levels on the chromatin of LIG1 null cells compared with the wild-type cells (Figure 6A, D and E). Despite olaparib being characterized as having intermediate to low PARP trapping activity (34), we observed robust retention of PARP2 on the chromatin fractions of LIG1 null cells starting at lower concentrations (100 nM) of olaparib as well as with shorter treatment times (Supplementary Figure S6A and B). As expected, expression of wild-type LigI in the LIG1 null cells prevented the increased retention of PARP2 (Supplementary Figure S7). Notably, the at least 4-fold increase in chromatin retention of PARP2 resulted in PARP2 being below the level of detection in the soluble fraction (Figure 6A, Supplementary Figure S6B and Supplementary Figure S7), indicating that, in the LIG1 null cells, the majority of PARP2 molecules are engaged in the joining of Okazaki fragments in the absence of LigI.

In accord with PARP1 being responsible for the majority of PAR synthesis (17,30,57), incubation with the PARP1 selective inhibitor AG14361 markedly reduced the steady state levels of PAR whereas the reported PARP2 selective inhibitor, UPF1069 did not (Figure 7). Furthermore, concordant with its lack of an effect on cell proliferation and survival (Supplementary Figure S5), UPF1069 also did not increase PARP2 trapping or reduce LigIIIα-XRCC1 association with chromatin (Figure 7). Interestingly, while the potent PARP1 selective inhibitor AG14361 (58) did not reduce LigIIIα-XRCC1 association with chromatin despite inhibiting PAR synthesis, it did increase the retention of PARP2 on the chromatin of LIG1 null cells (Figure 7). While the increased retention of PARP2 in LIG1 null cells could be due to AG14361 not being specific for PARP1, it is also possible that the increased retention of PARP2 could be due to the presence of PARP1 and/or inhibition of PAR synthesis by PARP1. To determine whether PARP1 is required for the increased retention of PARP2, we examined the effect of the PARP inhibitors in wild-type and LIG1 null cells lacking either PARP1 or PARP2. Notably, the steady state levels of chromatin-bound XRCC1 and LigIIIα were not reduced in untreated PARP1 null cells despite the lower levels of PAR (Figure 8A and Supplementary Figure S8) suggesting that either PAR synthesis by PARP2 is sufficient for the recruitment and retention of these proteins to chromatin containing endogenous DNA damage or there is a PAR-independent mechanism for their recruitment and retention. In the LIG1 null cells, the olaparib-induced retention of PARP2 was only slightly reduced in the absence of PARP1 (Figure 8A and B). Similar results were obtained in the wild-type and LIG1 null cells with AG14361 indicating that this PARP1 selective inhibitor also traps PARP2 on chromatin (Supplementary Figure S9). Thus, we conclude that the increased retention of PARP2 on chromatin caused by PARP inhibitors is independent of PARP1 and that AG14361 is not PARP1 specific.

Figure 7. Effect of PARP1 selective inhibitor, AG14361 on the chromatin binding of PARP1 and PARP2. Immunoblots of soluble and chromatin fractions obtained from CH12F3 wild-type cells (WT) and a LIG1 null derivative (LIG1−/−) incubated for 4 h with either DMSO (vehicle control) or AG14361 (1 μM) or UPF1069 (10 μM), with the indicated antibodies.

Figure 8. Chromatin retention of PARP2 by olaparib in the absence of PARP1. (A) Immunoblots of soluble and chromatin fractions obtained from CH12F3 LIG1 null (LIG1−/−) cells and derivatives lacking either PARP1 (PARP1−/−) or PARP2 (PARP2−/−) derivatives incubated for 4 h with either DMSO (vehicle control) or olaparib (10 μM), with the indicated antibodies. (B) Quantification of the steady state PARP2 levels in the chromatin fractions normalized to corresponding H3 levels. Data are the mean (±) SEM of three independent experiments. Unpaired two-tailed Student's t-test. ‘*’ P-values < 0.05.

Discussion

The replication of the lagging strand involves the co-ordinated action of multiple DNA replication proteins in a dynamic cyclical process (59–62). Notably, the DNA sliding clamp PCNA plays a critical central role through protein-protein interactions with DNA Pol δ, FEN1 and LigI (44,59,63–65). In addition to the interaction with PCNA that is critical for the recruitment of LigI to replication foci (66), LigI also physically and functionally interacts with RFC and Pol δ as well as the UHRF1 subunit of the DNA methylation machinery indicating that multiple protein-protein interactions with LigI contribute to Okazaki fragment joining and replication-associated DNA maintenance methylation (43,61,67). Given this network of interactions involving LigI, it is remarkable that LigIIIα-XRCC1 can effectively substitute for LigI (5) and that there are relatively few perturbations to the DNA replication machinery in the absence of LigI (9). Here we have examined the role of DNA damage-activated PARPs, PARP1 and PARP2, and PAR synthesis in the backup pathway for joining Okazaki fragments. Similar to LigI-deficient human cells (8), the LIG1 null mouse cells have elevated levels of PAR at replication foci. In accord with previous studies identifying PARP1 as a component of the DNA replication machinery (68,69), PARP1 associated with newly synthesized DNA and moved with the replication fork in both wild type and LIG1 null cells. Unexpectedly, higher levels of PARP2 were associated with the newly synthesized DNA in the LIG1 null cells compared with wild type cells with PARP2 also moving with the replication fork in the LIG1 null cells, suggesting that the increased association of PARP2 with newly synthesized DNA indicates a specific adaption to increase the activity of the backup pathway for joining Okazaki fragments to counter the defect in the canonical major pathway.

Previously it was shown that transient knockdown of PARP1 followed by LigI, reduced the plating efficiency of HeLa cells (7). As expected, given the apparent dependence of the backup pathway on PAR synthesis by PARP1 (7), the LIG1 null mouse cells were hypersensitive to the PARP1/2 inhibitor, olaparib. In contrast to the results with HeLa cells (7), knocking out PARP1 in the mouse LIG1 null cells did not significantly impact cell viability. Similarly, knocking out PARP2 in the mouse LIG1 null cells also did not significantly impact cell viability whereas loss of both PARP1 and PARP2 was synthetically lethal in the LIG1 null but not the wild type cells. Thus, we conclude that, in the mouse B cells lacking LigI, PAR synthesis by either PARP1 or PARP2 at the replication fork is sufficient for the recruitment of LigIIIα-XRCC1 to unligated Okazaki fragments and that this PAR-dependent recruitment for DNA replication is essential in the absence of LigI. Since PCNA unloading is likely to be coupled to ligation of Okazaki fragments (70) and its unloading from newly synthesized DNA was not reduced in the absence of LigI (9), it appears that the LigIIIα-XRCC1 complex is effectively able to join Okazaki fragments bound by PCNA. Interestingly, LigIIIβ, which lacks the C-terminal BRCT domain of LigIIIα, and consequently does not form a complex with XRCC1 (71,72), was unable to substitute for LigI in an in vitro reaction reconstituting Okazaki fragment synthesis, processing and ligation (61). XRCC1 interacts with PCNA (73) and so it is possible that this interaction contributes to the ability of the LigIIIα-XRCC1 complex to join unligated Okazaki fragments.

In contrast to the published studies that focused on PARP1 as a sensor of unligated Okazaki fragments (8,26), our studies showed a much higher increase in chromatin retention for PARP2 compared with PARP1 in the olaparib-treated LIG1 null cells. While the ability of olaparib to stabilize the binding of PARP2 but not PARP1 at SSBs likely contributes to the increased chromatin retention of PARP2 (20,34,39,42,74), the increased association of PARP2 but not PARP1 with newly synthesized DNA in the absence of a PARP inhibitor argues that PARP2 is preferentially recruited to ligatable SSBs on the lagging strand. Notably, in contrast to the relatively promiscuous strand break binding activity of PARP1 (75), PARP2 preferentially binds to single strand nicks with 3′ hydroxyl and 5′ phosphate termini (20), the final reaction intermediate generated by Okazaki fragment processing prior to ligation (61,62). Thus, our results are consistent with a model in which PARP1 moves with the replication machinery and acts as the sensor for unligated Okazaki fragments that occur infrequently in most cell types with a functional LigI-dependent canonical pathway whereas PARP2 plays a key role with the replication machinery when a larger number of unligated Okazaki fragments are generated, such as in LIG1 null cells.

Mice lacking PARP2 exhibit mild defects in erythropoiesis that do not occur in PARP1 knockout animals (76). Furthermore, it has been reported recently that homozygous knockin mice expressing PARP2 E343A, a catalytically dead version of PARP2 that binds stably to nicked DNA and blocks ligation, die in utero from anemia, and while the heterozygous mice are viable at birth, they exhibit more severe anemia than the PARP2 null mice (bioRxiv doi.org/10.1101/2024.03.12.584665). Notably, LIG1 null mice die at the same stage of embryogenesis as the homozygous parp2 knockin mice from a similar failure of erythropoiesis (3,4). During erythropoiesis, the switch from self-renewing stem cells to differentiation involves alterations in DNA replication that include a global increase in replication fork speed and a shorter S phase (77). Interestingly, erythrocytes from the homozygous knockin mice expressing PARP2 E343A exhibit high levels of replication-dependent genome instability, suggesting that the retention of catalytically dead PARP2 at unligated Okazaki fragments causes the failure of erythropoiesis (bioRxiv doi.org/10.1101/2024.03.12.584665). Thus, while it is likely that, in most cell types, the majority of Okazaki fragments are joined by the canonical LigI-dependent pathway, certain cell types, such as those involved in erythropoiesis, may be more sensitive to the loss of either PARP2 or LigI function because they are more dependent upon the backup pathway to join Okazaki fragments (3,4,76).

Since their initial FDA approval for the treatment of hereditary forms of ovarian cancer, the mechanisms of action of PARP inhibitors have been extensively studied and their use has been extended to many different cancer types (31–34,36,37,78). While the majority of currently available PARP inhibitors, including olaparib, are active against both PARP1 and PARP2 (79), their activity against homology-dependent repair defective cancers is dependent upon the presence of PARP1 (80,81) whereas the retention of PARP2 during DNA replication in specific cell types may be responsible for the side effects of PARP inhibitors that include anemia (76,79,82). This has prompted efforts to develop PARP1 specific inhibitors with reduced hematologic toxicities (79,83). Here, we were able to detect robust PARP2 trapping by the potent selective PARP1 inhibitor, AG14361, in the mouse LIG1 null cells despite its reported modest activity against PARP2 (58), indicating that measuring PARP2 chromatin retention in the LIG1 null cells may be a sensitive assay to assess PARP2 trapping by PARP1 selective inhibitors in cells that are dependent upon the backup pathway to join Okazaki fragments.

In summary, our studies have shown that PARP1 and PARP2 have critical overlapping roles in the LigIIIα-XRCC1 dependent backup pathway that is essential when the canonical LigI-dependent pathway for joining Okazaki fragment is defective. While PARP1 likely directs the infrequent LigIIIα-dependent joining of Okazaki fragments during unperturbed DNA replication in most cell types, the enrichment of PARP2 not PARP1 on newly replicated DNA and the increased trapping of PARP2 by PARP inhibitors in the absence of LigI in addition to the DNA substrate preference of PARP2 for unligated Okazaki fragments (20) suggest that PARP2 has a critical role in cell types that are dependent upon an increased contribution of the back-up pathway for DNA replication. This provides an explanation as to why certain cell types are more susceptible to PARP2 trapping. The presence of a robust LigI-independent backup pathway for joining Okazaki fragments has stimulated interest in LigI as a therapeutic target as this protein is frequently overexpressed in cancer (84) and, in ovarian cancer, high levels of LigI correlate with resistance to cisplatin and poor outcomes (85). While our study and others (7,8,26) predict that a combination of PARP1/2 and LigI inhibitors would be synthetically lethal in all replicating cells, HDR-deficient cells were hypersensitive to LigI inhibition (85), suggesting that LigI may be an alternative target to PARP1 in HDR-deficient cancers. Further work is needed to understand the effects caused by LigI inhibition compared with the absence of LigI.

Supplementary Material

gkae672_Supplemental_File

Acknowledgements

We thank Dr David Cortez and members of the Cortez laboratory for their advice and assistance with the aniPOND studies and the Vanderbilt Mass Spectrometry Research Center Proteomics Core for performing the mass spectrometry studies. We thank Drs Keifei Yu and Pierre Defossez for the CF12F3 cell lines. This study was supported by the Flow Cytometry and Bioinformatics Shared Resources in the University of New Mexico Comprehensive Cancer Center.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD039547.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

National Institutes of Health [R01 GM57479, R01 ES012512, R01 CA276837 to A.E.T., P01 CA92584 to P.I. Tainer]; University of New Mexico Cancer Center, an NCI-designated Comprehensive Cancer Center [CA118100 to P.I. Sanchez]. Funding for open access charge: NCI [CA276837].

Conflict of interest statement. None declared.

Notes

Present address: Nathaniel Wiest, Division of Hematology and Medical Oncology, Department of Internal Medicine, Mayo Clinic, Jacksonville, FL 32224, USA.

Present address: Ruofei Du, Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
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