Defective Repair of G₁-Phase Double-Strand Breaks in WRNIP1/Ku70 Double Knockout Cells

Abstract

Werner helicase-interacting protein 1 (WRNIP1) is a member of the AAA⁺ ATPase family and is conserved from Escherichia coli to Homo sapiens. Although a role for WRNIP1 in the S phase of the cell cycle has previously been reported, the present study demonstrates a novel function for this protein in G₁ phase. Deletion of WRNIP1 in non-homologous end joining (NHEJ)-deficient chicken DT40 cell lines resulted in slow growth, accumulation in G₁ phase, an increased population of dead cells, and accumulation of double-strand breaks (DSBs), which were indirectly evaluated by γH2AX. The data suggest that WRNIP1 prevents the generation of DSBs, executes DSB repair, and/or regulates DSB repair independently of NHEJ, particularly in G₁ phase. The potential molecular mechanism by which WRNIP1 functions in G₁ phase is discussed.

INTRODUCTION

Maintaining genome integrity is vital for cell viability, with various systems in place to support the completion of DNA replication and the repair of different DNA lesions. WRNIP1 is one of the proteins involved in these processes.

WRNIP1 belongs to the AAA⁺ ATPase family and is conserved from Escherichia coli to humans.¹⁾ It shares sequence homology with members of the replication factor C (RFC) family and has an ATPase domain featuring Walker A and B motifs, along with a ubiquitin-binding zinc-finger (UBZ) domain and two leucine-zipper motifs.^2–4) Our group and others have shown that WRNIP1 interacts physically or genetically with proteins involved in various cellular pathways. WRNIP1 interacts with components of the DNA replication machinery, including proliferating cell nuclear antigen (PCNA), replicative DNA polymerase δ (Polδ), and translesion synthesis (TLS) polymerase DNA polymerase η (Polη).^2,5,6) Monoubiquitinated PCNA promotes the replacement of Polδ with specialized TLS polymerases to allow replication to continue despite DNA lesions. We demonstrated that WRNIP1 is involved in regulation of Polη in TLS upon UV-induced damage.⁷⁾ Furthermore, upon DNA replication fork arrest, WRNIP1 could stabilize the arrested replication fork by recruiting Ataxia-telangiectasia mutated kinase (ATM) and promote resumption of DNA synthesis.⁸⁾ Thus, WRNIP1 could mainly act in S phase.

To know the functional relationship between WRNIP1 and ATM, we initially constructed WRNIP1/ATM double knockout (KO) chicken DT40 cells. The growth rate of these cells was slower than that of the single gene KO cells. ATM is a pivotal regulator of the DNA repair checkpoint and exerts influence over numerous pathways through the phosphorylation of target proteins. Upon detecting DNA double-strand breaks (DSBs), which pose a significant threat to the cell, ATM signals the presence of DNA damage and recruits other proteins to facilitate the repair process. Two major pathways exist in eukaryotic cells for the repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ).^9,10)

The HR pathway in somatic cells involves recombination between sister chromatids. Due to the dependence on the existence of identical copies of a post-replicative chromosome, HR is restricted to the S and G₂ phases, and results in accurate repair. NHEJ is a rapid and high-capacity pathway that joins two DNA ends with minimal reference to sequence, and the outcome is not necessarily an exact copy of the original DNA sequence. The pathway function is active throughout the cell cycle and plays a principal roles in the repair of DSBs in the absence of sister chromatids, such as during G₁ and the early S phases of the cell cycle. The NHEJ pathway involves Ku70-Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Previous research has indicated a role for ATM in the NHEJ pathway and the phosphorylation of DNA-PKcs, leading to the promotion of NHEJ.¹¹⁾

Considering the role of ATM in the NHEJ pathway, we proceeded to construct WRNIP1/DNAPKcs and WRNIP1/Ku70 double KO cells. While each single gene KO cell exhibited growth patterns comparable to those of wild-type cells, these double gene KO cells also grew slowly as WRNIP1/ATM cells. Further investigation of this slow growth phenotype led to the novel finding that in the absence of NHEJ pathway, WRNIP1 plays a role in DSB repair during the G₁ phase. The possible molecular mechanism by which WRNIP1 functions in DSB repair during the G₁ phase is discussed.

MATERIALS AND METHODS

Cell Culture and Reagents

DT40 cells were cultured at 39.5°C in D-MEM/Ham’s F-12 (FUJIFILM Wako, Osaka, Japan; 042-30555) supplemented with 100 µg/mL kanamycin, 10% fetal bovine serum, and 2% chicken serum.

Generation of WRNIP1^-/-/-/Parp1^-/-, WRNIP1^-/-/-/ATM^-/-\, WRNIP1^-/-/-/DNA-PKcs^-/-/-, and WRNIP1^-/-/-/Ku70^-/-Cells

Parp1^-/-,¹²⁾ ATM^-/-,¹³⁾ DNA-PKcs^-/-/-,¹⁴⁾ and Ku70^-/-14) cells were generated using chicken DT40 cells as described previously. WRNIP1 is located on chromosome 2, which is trisomic in DT40 cells. A GeneArt CRISPR Nuclease (CD4 Enrichment) Vector Kit (Thermo Fisher, Waltham, MA, U.S.A.; A21175) was used for the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) system. The target sequence for the WRNIP1 locus was designed to recognize 5′-CGAGGCGCGGCCTCTTGGAA-3′, annealed, and ligated into a linearized CRISPR/Cas9 vector to generate the WRNIP1-CRISPR/Cas9 vector. The WRNIP1-CRISPR/Cas9 vector and WRNIP1 KO vectors were transfected into Parp1^-/-, ATM^-/-, DNA-PKcs^-/-/-, and Ku70^-/- cells using the Neon Transfection System (Thermo Fisher) according to the manufacturer’s instructions. The primers used to confirm targeted integration are shown; WRNIP1 fw 5′-CTAGGCATGTGGCAGATAGAAAAACATAG-3′ WRNIP1 rv 5′-GGGTTACAAAACAAAATTGTAATGTTAG-3′. Chicken WRNIP1 expressing WRNIP1^-/-/- cells were generated by transfecting a previously used expression vector into WRNIP1^-/-/- cells.⁷⁾ The drug-resistant colonies have been acquired independently and meticulously cultivated to ensure their independency.

Drug Sensitivity Assays

Cells (1 × 10⁵) were treated with the ATM inhibitor Ku55933, the DNA-PK inhibitor Ku57788, the ATR inhibitor VE821, and Wortmannin (Funakoshi, Tokyo, Japan) at the specified concentrations and then cultured. The cell count was determined 24 h later.

Cell Cycle Assay

To determine the cell cycle phase, a Muse Cell Cycle Kit was used (CYTEK Biosciences, Fremont, CA, U.S.A.). Cells (1 × 10⁶) were centrifuged at 300 × g for 5 min, washed with phosphate-buffered saline (PBS), and resuspended in 50 µL of PBS. Thereafter, 1 mL of ice-cold 70% ethanol was slowly added to the resuspended cells. The cells were incubated at −20 °C for at least 3 h. Next, 200 µL of fixed cells was centrifuged at 300 × g for 5 min and washed with PBS. The cell pellet was resuspended in 200 µL of Muse Cell Cycle Reagent and incubated for 30 min at room temperature in the dark. The percentages of cells in G₀/G₁, S, and G₂/M phases of the cell cycle were analyzed with a Guava Muse Cell Analyzer.

Cell Death and γH2AX Assays

To evaluate cell death, 100 µL of Muse Annexin V & Dead Cell reagent (CYTEK Biosciences) was added to resuspended cells (1 × 10⁴/100 µL) and mixed. Samples were stained for 20 min at room temperature in the dark. The percentages of live, dead, early apoptotic, and late apoptotic cells were analyzed with a Guava Muse Cell Analyzer.

To measure histone H2AX phosphorylation, cells (1 × 10⁶) were centrifuged at 300 × g for 5 min, washed with PBS, and stained using a Muse H2A.X Activation Dual Detection Kit (CYTEK Biosciences). H2AX activation was analyzed with a Guava Muse Cell Analyzer.

Statistical Analysis

All data are represented as means ± standard error (S.E.). To evaluate statistical significance, Dunnett’s test was performed using the R statistical software version 4.2.2. A p-value less than 0.05 was considered to be statistically significant. All experiments were conducted at least three times.

RESULTS

Disruption of WRNIP1 Reduces Proliferation of ATM^-/- Cells

To gain insight into the function of WRNIP1 in the context of DNA damage, we sought to elucidate its relationship with factors involved in the DNA damage response. WRNIP1 is known to promote ATM signaling in response to replication stress.⁸⁾ To know the functional relationship between WRNIP1 and ATM further, we constructed WRNIP1^-/-/-/ATM^-/- cells by targeting ATM^-/- cells with a guide RNA CRISPER/Cas9 vector along with the WRNIP1 targeting construct.

Disruption of corresponding genes was confirmed by RT-PCR (Fig. 1A, left). The impact of WRNIP1 disruption on proliferation of ATM^-/- cells was evaluated. WRNIP1 disruption resulted in a slight delay in the growth of ATM^-/- cells (Fig. 1A, right).

Fig. 1. Effect of WRNIP1 Disruption on Proliferation of ATM^-/- Cells

(A) Generation of WRNIP1^-/-/-/ATM^-/- cells. Confirmation of disruption of WRNIP1 and ATM by RT-PCR. Growth curve. Cells (1 × 10⁵) were inoculated in 1 mL of medium and cultured for the indicated durations. The number of viable cells was measured every 24 h using the trypan blue dye exclusion method. All values represent the mean ± standard error (S.E.) from four experiments. ^***p < 0.001. (B) The effect of Wortmannin addition on the survival rate of WRNIP1-deficient cells. Wortmannin was added, and the cell survival rate was measured 2 days later. All data points are shown in circles and the vertical bars represent the mean ± S.E. from four experiments. (C) Cell proliferation ability of WRNIP1-deficient cells upon Wortmannin addition. Wild-type, WRNIP1-deficient cells and chicken WRNIP1expressing WRNIP1-deficient cells (1 × 10⁵) were inoculated in 1 mL of medium to which Wortmannin was added to achieve a final concentration of 320 nM, and they were cultured for the indicated durations. The number of viable cells was measured every 24 h using the trypan blue dye exclusion method. The symbols indicate the mean ± S.E. of three experiments. ^***p < 0.001.

In light of the unanticipated slow growth of WRNIP1^-/-/-/ATM^-/- cells, we investigated the impact of ATM inhibitors on WRNIP1^-/-/- cells. Wortmannin is a selective inhibitor of PI3K family members, including ATM and DNA-PK.¹⁵⁾ WRNIP1^-/-/- cells were more sensitive to Wortmannin than wild-type (WT) cells (Fig. 1B). Following addition of Wortmannin, the growth of WRNIP1^-/-/- cells was delayed compared with that of WT cells, indicating that in the absence of ATM function, WRNIP1 plays an important role for the growth of the cells (Fig. 1C). In order to provide further confirmation of this result, an expression vector carrying chicken WRNIP1 cDNA was introduced into WRNIP1^-/-/- cells. The presence of wild-type WRNIP1 protein successfully restored the Wortmannin sensitivity of WRNIP1^-/-/- cells (Fig. 1C).

WRNIP1 KO Cells Grow Slowly upon Disruption of Genes Involved in NHEJ Pathway

We examined the sensitivity of WRNIP1^-/-/- cells to other inhibitors of phosphatidylinositol-3 kinase (PI3K) family members. WRNIP1^-/-/- cells were more sensitive to Ku55933 (ATM inhibitor), Wortmannin (ATM and DNA-PK inhibitor), and Ku57788 (DNA-PK inhibitor) than WT cells. Conversely, the sensitivity of WRNIP1^-/-/- cells to the ATR inhibitor VE821 was comparable with that of WT cells (Fig. 2A).

Fig. 2. Effect of WRNIP1 Disruption on Proliferation of DNA-PKcs^-/-/- Cells and Ku70^-/- Cells

(A) Cell survival rate upon addition of each drug. Drugs were added to WT and WRNIP1^-/-/- cells, and the number of viable cells was measured 24 h later. All data points are shown in circles and the vertical bars represent the mean ± S.E. from four experiments. ^***p < 0.001. (B) Generation of WRNIP1^-/-/-/DNA-PKcs^-/-/-cells. Confirmation of disruption of WRNIP1 and DNA-PKcs by RT-PCR. Growth curve. Cells (1 × 10⁵) were inoculated in 1 mL of medium and cultured for the indicated durations. The number of viable cells was measured every 24 h using the trypan blue dye exclusion method. The symbols indicate the mean ± S.E. of three experiments. ^***p < 0.001. (C) Generation of WRNIP1^-/-/-/Ku70^-/-cells. Confirmation of disruption of WRNIP1 and Ku70 by RT-PCR. Growth curve. Cells (1 × 10⁵) were inoculated in 1 mL of medium and cultured for the indicated durations. The number of viable cells was measured every 24 h using the trypan blue dye exclusion method. The symbols indicate the mean ± S.E. of three experiments. ^***p < 0.001.

ATM was shown to phosphorylate DNA-PKcs and activate its activity, resulting in the promotion of NHEJ.¹¹⁾ Given the crucial role of the protein phosphorylation activity of DNA-PK in efficient NHEJ of DSBs,¹⁶⁾ it is plausible that Ku55933, Wortmannin, and Ku57788 impact the NHEJ pathway, resulting in slow growth of WRNIP1^-/-/-cells. To test this hypothesis, we generated WRNIP1^-/-/-/DNA-PKcs^-/-/- cells by co-transfecting the constructs into previously generated DNA-PKcs^-/-/- cells. DNA-PKcs is the catalytic subunit of DNA-PK. Two clones were obtained, both of which exhibited disruption of WRNIP1, as confirmed by RT-PCR (Fig. 2B, left). During cell culture, one clone demonstrated immediate recovery of proliferation, potentially due to acquisition of additional mutations. Consequently, the remaining clone was analyzed.

Proliferation of WRNIP1^-/-/-/DNA-PKcs^-/-/- cells was significantly lower than that of both single KO cell lines, indicating that NHEJ is crucial for normal proliferation of WRNIP1^-/-/- cells (Fig. 2B, right). If this possibility is correct, disruption of Ku70, another component of NHEJ pathway in WRNIP1^-/-/- cells will result in slow growth.

WRNIP1^-/-/-/Ku70^-/- cells were generated and disruption of both WRNIP1 and Ku70 was confirmed by RT-PCR (Fig. 2C, left). Proliferation of WRNIP1^-/-/-/Ku70^-/- cells was lower than that of both single KO cell lines (Fig. 2C, right). The introduction of chicken WRNIP1 cDNA into WRNIP1^-/-/-/Ku70^-/- cells resulted in restoration of the slow growth phenotype exhibited by the double mutant, as illustrated in Supplementary Fig. 2. We conclude that NHEJ plays a significant role in proliferation of WRNIP1 KO cells.

WRNIP1-Disrupted NHEJ-Deficient Cells Accumulate in G₁ Phase

Almost all functions of WRNIP1 so far reported are concerned with events occurring in the S phase and the NHEJ pathway is active throughout the cell cycle, although it becomes important in the G₁ to early S phase (see Introduction). Therefore, we expected WRNIP1^-/-/-/DNA-PKcs^-/-/- and WRNIP1^-/-/-/Ku70^-/- cells to accumulate in the S phase. Surprisingly, both WRNIP1^-/-/-/DNA-PKcs^-/-/- (Fig. 3A) and WRNIP1^-/-/-/Ku70 ^-/- (Fig. 3B) cells significantly accumulated in the G₁ phase. The accumulation of WRNIP1^-/-/-/DNA-PKcs^-/-/- and WRNIP1^-/-/-/Ku70^-/- cells in G₁ phase suggests that unrepaired DSBs due to the defects in the NHEJ pathway trigger the G₁ checkpoint.

Fig. 3. Effect of WRNIP1 Disruption on the Cell Cycle Distribution of NHEJ-Disrupted Cells

(A) WRNIP1^-/-/-/DNA-PKcs^-/-/- cells. (B) WRNIP1^-/-/-/Ku70^-/- cells. The percentages of cells in G₀/G₁, S, and G₂/M phases of the cell cycle were measured with a Guava Muse Cell Analyzer using a Muse Cell Cycle Kit. All data points are shown in circles and the vertical bars represent the mean ± S.E. from five experiments (A). ^**p < 0.01. All data points are shown in circles and the vertical bars represent the mean ± S.E. from seven experiments (B). ^***p < 0.001.

Noteworthy, NHEJ plays a pivotal role in DSB repair at the G₀/G₁ stage, as evidenced by the increased radiosensitivity of cells lacking NHEJ-related proteins at this stage.¹⁴⁾ The proliferative properties of DT40 KU70^-/-, DNA-PKcs^-/-/-, and KU70^-/-/DNA-PKcs^-/-/- cells were found to be indistinguishable from those of wild-type cells, as monitored by growth curves and by cell-cycle analysis.¹⁴⁾ Consequently, there had been no data on the accumulation of NHEJ-deficient cells at the G₁ stage until now. Thus, this study will be the first to report the effects of WRNIP1 and NHEJ-related defects on the G₁ phase.

DSBs Accumulate in WRNIP1/Ku70 Double KO Cells

In order to verify the presence of DSBs in WRNIP1^-/-/-/Ku70^-/- cells, we closely monitored the levels of γH2AX (phosphorylated form of H2AX), as this has been shown to be an excellent marker of DSBs.¹⁷⁾ ATM, ATR, and DNA-PKcs are the kinases responsible for phosphorylating H2AX in the presence of DSBs. Therefore, γH2AX-positive cells can be counted upon to indirectly monitor accumulation of DSBs in cells. The percentage of γH2AX-positive cells was higher among WRNIP1^-/-/-/Ku70^-/- cells than among WT cells (Fig. 4A), indicating that DSBs accumulate in WRNIP1^-/-/-/Ku70^-/- cells.

Fig. 4. Effect of WRNIP1 Disruption on DSB Formation and Viability of Ku70^-/- Cells

(A) Detection of γH2AX-positive cell. Upon generation of DSBs, H2AX is phosphorylated at serine 139 (forming γH2AX) by ATM. This phosphorylation marks the site of damage and serves as a signal for recruitment of other DNA repair proteins to the break site. Levels of histone H2AX phosphorylation were measured as a means of indirectly gauging the presence of DSBs. This was achieved by utilizing an anti-Histone H2A.X antibody and a phospho-specific anti-phospho-Histone H2A.X (Ser139) antibody. This approach enabled the measurement of both total and phosphorylated proteins within a single cell, thereby providing a comprehensive assessment of the phosphorylation level of H2A.X. All data points are shown in circles and the vertical bars represent the mean ± S.E. from four experiments. ^**p < 0.01. (B) Quantitative analysis of live, early apoptotic, late apoptotic, and dead cells. Apoptosis is characterized by the translocation of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane, leading to its exposure on the extracellular surface. By combining the dead cell marker 7-AAD with fluorescently labeled Annexin V, we detected phosphatidylserine on the surface of apoptotic cells. In the early stages of apoptosis, phosphatidylserine translocates to the outer surface of the cell membrane, where it can bind to Annexin V. As the process progresses, membrane integrity is compromised, and the membrane-impermeable dye 7-AAD is used to distinguish dead cells from early apoptotic cells. This assay enables the categorization of four populations: live cells without apoptosis, early apoptotic cells, late apoptotic cells, and dead cells not involving the apoptotic pathway. All data points are shown in circles and the vertical bars represent the mean ± S.E. from four experiments. ^***p < 0.001.

Although apoptosis was not increased in WRNIP1^-/-/-/Ku70^-/- cells compared with WT cells and the corresponding single KO cell lines, the number of dead cells was increased among WRNIP1^-/-/-/Ku70^-/- cells (Fig. 4B). Thus, we conclude that accumulation of DSBs in WRNIP1- and NHEJ-deficient cells triggers accumulation in the G₁ phase and an increase of dead cells, leading to their slow growth.

DISCUSSION

Many of the functions of WRNIP1 so far reported are somehow concerned with events in the S phase.¹⁸⁾ For instance, WRNIP1 has been shown to bind to forked DNA that resembles stalled forks,¹⁹⁾ protect stalled forks from degradation,^20,21) and promote fork restart upon replication stress.^20,21) Furthermore, it has been demonstrated to control translesion synthesis.⁷⁾ In contrast, the data obtained with WRNIP1 and NHEJ pathway gene double KO cells that contain DSBs and accumulate in G₁ phase suggest that, in the absence of the NHEJ pathway, WRNIP1 plays an important role in the G₁ phase.

With regard to the role of WRNIP1 in the G₁ phase, two possibilities can be considered. First, WRNIP1 is involved in the process of decreasing the formation of DSBs. Second, WRNIP1 is involved in the process of repairing DSBs. WRNIP1 prevents transcription-associated genomic instability by regulating R-loop formation.²²⁾ However, the genomic instability is caused by the collision between the transcription machinery and the replisome that occurs in the S phase. This scenario does not explain the role played by WRNIP1 in the G₁ phase.

With regard to DSB repair, there are several pathways in cells in addition to NHEJ. These are HR, single-strand annealing (SSA), and alternative end joining (a-EJ), also known as microhomology-mediated end joining.²³⁾ As repair by HR and SSA is favored in the S and G₂ phases, it seems likely that in a cell defective in NHEJ, the pathway working in G₁ phase is a-EJ. This pathway involves DNA polymerase θ (Polθ) and poly(ADP-ribose) polymerase 1 (PARP1). It is interesting to note that WRNIP1^-/-/-/Parp1^-/- double KO cells grew almost similarly to both single KO cell lines (Supplementary Fig. 1), suggesting that WRNIP1 and PARP1 function in the same pathway. In addition to our previous finding that WRNIP1 interacts with Polδ and Polη, recent reports state that WRN and WRNIP1, along with Rev1 (a TLS polymerase), are required for TLS by Y family polymerases, including Polη, Polι, and Polκ.²⁴⁾ Furthermore, previous analyses have demonstrated that WRNIP1 rapidly accumulates at laser-irradiated sites where DSBs exist.²⁵⁾ Consequently, it can be hypothesized that WRNIP1 is involved in the a-EJ pathway in conjunction with PARP1, playing a role by interacting functionally with Polθ. The relationship between WRNIP1 and Polθ will be investigated in future studies.

Acknowledgments

We thank Dr. Takemi Enomoto for criticism and comments on our first manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Kawabe Y, Branzei D, Hayashi T, Suzuki H, Masuko T, Onoda F, Heo SJ, Ikeda H, Shimamoto A, Furuichi Y, Seki M, Enomoto T. A novel protein interacts with the Werner’s syndrome gene product physically and functionally. J. Biol. Chem., 276, 20364–20369 (2001).
• 2)  Tsurimoto T, Shinozaki A, Yano M, Seki M, Enomoto T. Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase δ. Genes Cells, 10, 13–22 (2005).
• 3)  Bish RA, Myers MP. Werner helicase-interacting protein 1 binds polyubiquitin via its zinc finger domain. J. Biol. Chem., 282, 23184–23193 (2007).
• 4)  Crosetto N, Bienko M, Hibbert RG, Perica T, Ambrogio C, Kensche T, Hofmann K, Sixma TK, Dikic I. Human Wrnip1 is localized in replication factories in a ubiquitin-binding zinc finger-dependent manner. J. Biol. Chem., 283, 35173–35185 (2008).
• 5)  Saugar I, Parker JL, Zhao S, Ulrich HD. The genome maintenance factor Mgs1 is targeted to sites of replication stress by ubiquitylated PCNA. Nucleic Acids Res., 40, 245–257 (2012).
• 6)  Yuasa MS, Masutani C, Hirano A, Cohn MA, Yamaizumi M, Nakatani Y, Hanaoka F. A human DNA polymerase η complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin-bound fraction of cells undergoing replication fork arrest. Genes Cells, 11, 731–744 (2006).
• 7)  Yoshimura A, Kobayashi Y, Tada S, Seki M, Enomoto T. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response. Biochem. Biophys. Res. Commun., 452, 48–52 (2014).
• 8)  Kanu N, Zhang T, Burrell RA, Chakraborty A, Cronshaw J, DaCosta C, Grönroos E, Pemberton HN, Anderton E, Gonzalez L, Sabbioneda S, Ulrich HD, Swanton C, Behrens A. RAD18, WRNIP1 and ATMIN promote ATM signalling in response to replication stress. Oncogene, 35, 4009–4019 (2016).
• 9)  Ceccaldi R, Rondinelli B, D’Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol., 26, 52–64 (2016).
• 10)  Scully R, Panday A, Elango R, Willis NA. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat. Rev. Mol. Cell Biol., 20, 698–714 (2019).
• 11)  Lu H, Zhang Q, Laverty DJ, Puncheon AC, Augustine MM, Williams GJ, Nagel ZD, Chen BPC, Davis AJ. ATM phosphorylates the FATC domain of DNA-PK_cs at threonine 4102 to promote non-homologous end joining. Nucleic Acids Res., 51, 6770–6783 (2023).
• 12)  Hochegger H, Dejsuphong D, Fukushima T, Morrison C, Sonoda E, Schreiber V, Zhao GY, Saberi A, Masutani M, Adachi N, Koyama H, de Murcia G, Takeda S. Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells. EMBO J., 25, 1305–1314 (2006).
• 13)  Takao N, Kato H, Mori R, Morrison C, Sonada E, Sun X, Shimizu H, Yoshioka K, Takeda S, Yamamoto K. Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene, 18, 7002–7009 (1999).
• 14)  Fukushima T, Takata M, Morrison C, Araki R, Fujimori A, Abe M, Tatsumi K, Jasin M, Dhar PK, Sonoda E, Chiba T, Takeda S. Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G₂ phase DNA double-strand break repair. J. Biol. Chem., 276, 44413–44418 (2001).
• 15)  Matsumoto Y. Development and evolution of DNA-dependent protein kinase inhibitors toward cancer therapy. Int. J. Mol. Sci., 23, 4264 (2022).
• 16)  Yue X, Bai C, Xie D, Ma T, Zhou PK. DNA-PKcs: a multi-faceted player in DNA damage response. Front. Genet., 11, 607428 (2020).
• 17)  Raavi V, Perumal V, Paul SFD. Potential application of γ-H2AX as a biodosimetry tool for radiation triage. Mutat. Res. Rev. Mutat. Res., 787, 108350 (2021).
• 18)  Yoshimura A, Seki M, Enomoto T. The role of WRNIP1 in genome maintenance. Cell Cycle, 16, 515–521 (2017).
• 19)  Yoshimura A, Seki M, Kanamori M, Tateishi S, Tsurimoto T, Tada S, Enomoto T. Physical and functional interaction between WRNIP1 and RAD18. Genes Genet. Syst., 84, 171–178 (2009).
• 20)  Leuzzi G, Marabitti V, Pichierri P, Franchitto A. WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress. EMBO J., 35, 1437–1451 (2016).
• 21)  Porebski B, Wild S, Kummer S, Scaglione S, Gaillard PL, Gari K. WRNIP1 protects reversed DNA replication forks from SLX4-dependent nucleolytic cleavage. iScience, 21, 31–41 (2019).
• 22)  Marabitti V, Lillo G, Malacaria E, Palermo V, Pichierri P, Franchitto A. Checkpoint defects elicit a WRNIP1-mediated response to counteract R-loop-associated genomic instability. Cancers (Basel), 12, 389 (2020).
• 23)  Chang HHY, Pannunzio NR, Adachi N, Lieber MR. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat. Rev. Mol. Cell Biol., 18, 495–506 (2017).
• 24)  Yoon J-H, Sellamuthu K, Prakash L, Prakash S. WRN exonuclease imparts high fidelity on translesion synthesis by Y family DNA polymerases. Genes Dev., 38, 213–232 (2024).
• 25)  Nomura H, Yoshimura A, Edo T, Kanno S, Tada S, Seki M, Yasui A, Enomoto T. WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain. Biochem. Biophys. Res. Commun., 417, 1145–1150 (2012).