Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation.


Introduction
The DNA damage response (DDR) is a surveillance system evolved to safeguard genome integrity in the face of genotoxic stress throughout an organism's life.Upon DNA damage, a complex signaling pathway activates the DDR to coordinate the detection of DNA lesions (checkpoint activation), cell cycle arrest, and efficient DNA repair [1].Following successful repair, DDR activates checkpoint recovery to resume cell cycle progression [2,3].One of the most severe forms of DNA damage is double-stranded breaks (DSBs), which require immediate repair through processes such as homologous recombination (HR) or non-homologous end joining (NHEJ) [4].Dysregulation of these processes may produce gene mutations, chromosome aberration, and aneuploidy [5], hallmarks of proliferative diseases and genetic disorders [6,7].Therefore, precise control and fidelity are crucial during the repair process.
Reversible protein phosphorylation is a highly conserved molecular mechanism in eukaryotes that orchestrates complex cellular processes, including cell cycle progression and DNA damage response [8,9].Phosphorylation is catalyzed by protein kinases and reversed by protein phosphatases, and serves as a molecular switch, modulating the activity, structure, half-life, localization, and interacting partners of target proteins.Checkpoint activation and DNA repair are mainly regulated by the extensively studied master kinases, the Ataxia-telangiectasia and Rad3-related (ATR), Ataxia-telangiectasia mutated (ATM), and DNA-dependent protein kinases (DNA-PK) [10], as well as their effectors, Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2/Radiation-sensitive 53 (Chk2/RAD53, mammal/yeast nomenclature) [11].However, dynamic phosphorylation and dephosphorylation of DDR regulators and executor molecules are critical for successful DNA repair and maintenance of genome integrity.It has become evident over the past years that several protein phosphatases that counteract the activity of DDR kinases and dephosphorylate their substrates are involved in this process.The five major phosphatases that contribute directly to DDR regulation are the Ser/Thr Phosphoprotein phosphatase 1 (PPP1), Phosphoprotein phosphatase 2A (PPP2A), Phosphoprotein phosphatase 2C/WIP1 and Phosphoprotein phosphatase 4 (PPP4, hereafter PP4), as well as Cdc14, which is a dual specificity enzyme [12,13].
Centrobin (CNTRB) was initially identified as a centrosomal protein that interacts with BRCA2 in human cells [32].It plays a vital role in daughter centriole duplication and elongation [32,33], and contributes to microtubule stability and dynamics, regulated by NEK2 and polo-like kinase 1 [34][35][36][37][38]. CNTRB has been identified as a putative substrate of ATM/ATR kinases, which recognize and phosphorylate specific motifs, such as serineglutamine (SQ) or threonine-glutamine (TQ) [39].Recent studies have demonstrated that ATR kinase specifically phosphorylates CNTRB in response to UV-induced DNA damage, leading to its enrichment in the nuclear matrix.This modification is essential for cell survival and proper DNA repair through HR [40].However, the identity of the phosphatase responsible for dephosphorylating CNTRB in DDR remains unknown.We note that the previously identified PP4-binding motif in human CNTRB (771-FRVP-774 [29]) is in close proximity to the putative ATR-targeted SQ motif of CNTRB (781-SQ-782, Figure 1a).In addition, it is known that PP4 interacts with the ATR-interacting protein, ATRIP and, together with ATR, they co-regulate the phospho-status of different substrates [22].This leads to the speculation that PP4 may play a role in controlling the function of CNTRB in DNA repair.
In this study, we provide evidence for the physical interaction between PP4 and CNTRB.We also show that the integrity of the FRVP and SQ motifs in CNTRB is crucial for repairing irradiation-induced double-stranded DNA breaks through HR in human cells.Exclusion of PP4 from CNTRB or mutation of the SQ motif results in a significant delay in HR and the accumulation of chromosomes with abnormal morphology, likely due to defective Holliday junction resolution [5,41].Our findings imply a novel function of PP4 in the late stages of HR, and further support the role of PP4 in facilitating cell cycle resumption following DNA repair.
Total protein extracts were made by boiling the cells in Laemmli sample buffer for 5 min; then, 10 µg of each protein sample was run on SDS-PAGE followed by immunoblotting using anti-CNTRB, anti-PP4c, anti-R3A, anti-R3B, and anti-αTubulin antibodies, respectively.
Total RNA purification was performed with Quick-RNA MiniPrep kit (cat#R1054, Zymo Research, Irvine, CA, USA).For the synthesis of first-strand cDNA, the RevertAid First Strand cDNA Synthesis Kit (cat#K1670, Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer's instructions.Maxima SYBR Green/ROX qPCR Master Mix (cat#K0222, Thermo Fisher Scientific, Waltham, MA, USA) was used for the realtime quantitative PCR reaction, according to the manufacturer's instructions.Reactions were run three times in quadruplicates in the Rotor-Gene Q Real-Time PCR Detection System (QIAGEN, Germantown, MD, USA) with the following reaction conditions: 95 • C 10 min, 40 cycles of 95 • C 15 s, 55 • C 30 s, 72 • C 30 s.The final values represent the mean and standard error of the quadruplicates.

γH2AX Foci Quantitation Assay
HeLa cells were cultivated in 6-well tissue culture plates (cat#30006, SPL Life Sciences, Pochon, Kyonggi-do, South Korea), and following 24 h of gene silencing, the cells were trypsinized (cat#TRY-3B, Capricorn Scientific, Ebsdorfergrund, Germany) using standard protocols and transferred to glass-bottom cell culture chambers (cat#631-0150, VWR International, Radnor, PA, USA).Twenty-four hours post gene silencing, the cells were exposed to 2 Gy X-rays using a Trakis XR-11 X-ray machine according to previous work [46].At various time points after irradiation, the cells were fixed with 3% PFA (paraformaldehyde, cat#158127, Merck Millipore, Burlington, MA, USA) for 10 min and subjected to immunofluorescence.The γH2AX foci were quantified in 50 EdU-positive S-phase cells after anti-γH2AX immunostaining.The remaining cells were collected for qPCR analysis.

Statistical Analysis of Data
All experiments were replicated at least three times.Statistical analysis was performed using Origin Graph statistical software (https://www.originlab.com/origin(accessed between 1 March 2023 and 1 August 2023)).In all cases, we made an ANOVA test [47].Asterisks represent p values, which correspond to the significance of regression coefficients (* p < 0.05, ** p < 0.01, and *** p < 0.001).

DR-GFP Reporter Assay
DR-GFP U2OS cells, obtained from Jeremy Stark [45], were initially seeded in 6-well tissue culture plates, followed by the silencing of target genes.After 24 h, the cells were trypsinized and transferred to glass-bottom cell culture chambers.Subsequently, 24 h post-transfection, the cells were transfected with an I-SceI rare cutter endonuclease coding plasmid construct (cat#26477, Addgene Watertown, MA, USA).After 96 h of gene silencing, the cells were fixed with 3% PFA for 10 min.The cells were then visualized using confocal microscopy, and the number of GFP-positive cells, indicative of homologous recombination (HR) events, was quantified using CellProfiler software (version 4.2.6).In CellProfiler, the nuclei were segmented based on Hoechst-stained DNA.A minimum of 3000 cells were analyzed per experimental condition (Table S2).

Chromosome Preparation
HeLa cells were seeded in 6-well tissue culture plates and transfected with siRNAs, or co-transfected with siRNAs and flag constructs.After 24 h, cells were separated into two 6-well plates, one was irradiated with X-ray using Trakins XR-11 X-ray machine, then treated with 20 µg/mL caffeine (cat#C0750, Merck Millipore, Burlington, MA, USA) and 10 µg/mL colchicine (cat#D00138122, Calbiochem, San Diego, CA, USA), then incubated for 6 h at 37 • C at 5% CO 2 .Cells were trypsinized and harvested with centrifugation (1000× g, RT, 3 min) and incubated for 30 min at 37 • C with 75 mM KCl.Cells were washed three times with ice-cold methanol and acetic acid in a 3:1 ratio, 20 µL was then dried onto coverslips and stained with Hoechst (1 µg/mL in PBS) before 150 chromosome spreads were counted and classified per sample.

Double Transfection of siRNAs and Transgenic Constructs
Hela cells were initially plated in a 6-well tissue culture plate and co-transfected with siControl and Flag vector, or siCNTRB along with Flag vector, or wild-type (WT), S781A, S781D or ARVA variants of Flag-CNTRB 180-903aa .After 24 h, the cells were divided into two tissue culture plates.At 48 h post-transfection, half of the cells were subjected to X-ray treatment.Following X-ray treatment, the cells were treated with 20 µg/mL caffeine and 10 µg/mL colchicine and incubated for 6 h at 37 • C in a 5% CO 2 environment.Chromosomes were prepared as described above.

Immunofluorescence and Microscopy
Twenty-four hours post-transfection, cells were seeded onto coverslips, incubated for another 24 h followed by fixation with 3% PFA for a duration of 10 min.Subsequently, the cells were permeabilized using 0.5% Triton X-100 in PBS for 10 min, then washed three times with PBS.To block non-specific binding, the cells were incubated in a blocking buffer containing 3% bovine serum albumin and 0.1% Triton X-100 in PBS for 1 h at room temperature (RT).The samples were then treated with primary antibody diluted in blocking buffer overnight at 4 • C.After incubation with the primary antibody, the cells were washed three times with 0.1% Triton X-100 in PBS and incubated with a fluorescently tagged secondary antibody diluted in blocking buffer for 1 h at RT in the dark.Following two washes with 0.1% Triton X-100 in PBS, the cells were counterstained with Hoechst (1 µg/mL in PBS) for 10 min.Microscopy images were captured using a Zeiss LSM800 system.

Apoptosis Assay
HeLa cells were grown in 6-well tissue culture plates and transfected with gene-specific siRNAs to silence cntrob, pp4c, r3a or r3b, respectively.At 48 h post-transfection, cells were exposed to 2 Gy X-ray irradiation.At 8 h post-irradiation, cells were trypsinized, fixed with 70% ethanol on ice, and DNA was stained with 50 µg/mL propidium iodide (cat#P4170, Sigma) solution for 30 min followed by flow cytometric analysis (CytoFlexBecton, Dickinson and Company Biosciences, San Jose, CA, USA).

The R3 Subunit of PP4 Directly Binds to CNTRB through Its FRVP Motif
It has been reported that the carboxy-terminal half of human CNTRB (CNTRB 460-903aa ), which contains a genuine PP4-binding motif (771-FRVP-774, Figure 1a), specifically interacts with both isoforms of R3 in HeLa cells [29].Therefore, it was critical to understand whether the binding of full-length CNTRB to R3A or R3B depends exclusively on this single FRVP motif or if additional sequences are also needed for the interaction.To test this, we performed an in vitro pull-down assay and found that immobilized recombinant GST-R3A and GST-R3B specifically bind to the 35 S-methionine-labeled wild-type CNTRB produced in in vitro-coupled transcription and translation reactions (IVTT).However, when we mutated phenylalanine and proline to alanine in the FRVP motif (FRVP to ARVA), the interaction was completely abolished (Figure 1b).This indicates that CNTRB has a single PP4-binding motif and that FRVP is necessary and sufficient for R3A/R3B recruitment.
We encountered difficulties when we wanted to validate the in vitro binding results with in vivo experiments using full-length CNTRB.We (Figure S1), and others [32], have found that overexpressed CNTRB forms aggregates around the nucleus and on the cytoskeleton and does not localize to the centrosome.Moreover, we observed the same when the amino-terminal (CNTRB 1-460aa ) or carboxy-terminal (CNTRB 460-903aa ) [29] halves of CNTRB were overexpressed separately in human cells (Supplementary Figure S1).This may be due to the otherwise low levels of endogenous CNTRB, which is tightly regulated, as well as due to interrupted structural elements in the truncated overexpressed proteins (Figure S1a).Therefore, based on secondary structure prediction, we designed a transgenic fragment of CNTRB lacking the first 180 amino acids (hereafter CNTRB 180-903aa ).When overexpressed, CNTRB 180-903aa was soluble and showed centrosomal localization in human cells (Figure S1b,c).Therefore, we co-expressed flag-tagged CNTRB 180-903aa and its PP4-binding-deficient mutant, ARVA, together with GFP, GFP-R3A, or GFP-R3B, respectively, in HEK293 cells.Using co-immunoprecipitation experiments, we demonstrated that both GFP-R3A and GFP-R3B bind to CNTRB 180-903aa , which requires an intact FRVP motif (Figure 1c).All these results imply that PP4 binds specifically to a single motif in CNTRB 180-903aa .In addition, we proved that CNTRB 180-903aa is a soluble centrosomal protein, with the potential for use in functional assays.

CNTRB and PP4 Co-Operate in DNA Damage Repair
The variant of histone H2AX undergoes rapid phosphorylation at serine 139 (referred to as γH2AX [48]) in the microenvironment of chromatin surrounding DSBs [49].This modification facilitates the accumulation of DNA repair machinery [50] and acts as a signal for DNA damage by activating other DNA repair factors [51].As mentioned earlier, PP4 is responsible for dephosphorylation of γH2AX, which is necessary to restore the normal progression of the cell cycle after DNA damage has been repaired [20].To explore the link between CNTRB and PP4 during DNA repair in the S-phase, we utilized EdU (5-ethynyl 2 -deoxyuridine) pulse-labeled HeLa cells and compared the dynamics of γH2AX in cells silenced for cntrob, pp4c, r3a, r3b, or cntrob and pp4c, respectively, after X-ray irradiation.We quantified the downregulation of the indicated genes by western blotting and qPCR (Figure S2e,f and Table S1).All conditions showed a significant increase in the number of γH2AX nuclear foci compared to the control sample, indicating a synergistic connection between CNTRB and PP4 function during the DNA repair process (Figure 2a and Table S2).Sustained levels of γH2AX 8 h after irradiation suggest a strong delay in DNA repair (Figures 2a and S2a-d).To validate these results, we repeated the experiment with a second set of siRNAs with different target sequences and found a similar phenotypic change in cells (Figure S2g-i and Table S2).Simultaneously, we quantified the downregulation of the indicated genes by qPCR, normalized to actin control (Figure S2j and Table S1).We encountered difficulties when we wanted to validate the in vitro binding results with in vivo experiments using full-length CNTRB.We (Figure S1), and others [32], have found that overexpressed CNTRB forms aggregates around the nucleus and on the cytoskeleton and does not localize to the centrosome.Moreover, we observed the same when the amino-terminal (CNTRB 1-460aa ) or carboxy-terminal (CNTRB 460-903aa ) [29] halves of CNTRB were overexpressed separately in human cells (Supplementary Figure S1).This may be due to the otherwise low levels of endogenous CNTRB, which is tightly regulated, as well as due to interrupted structural elements in the truncated overexpressed proteins (Figure S1a).Therefore, based on secondary structure prediction, we designed a transgenic fragment of CNTRB lacking the first 180 amino acids (hereafter CNTRB 180-903aa ).When overexpressed, CNTRB 180-903aa was soluble and showed centrosomal localization in human cells (Figure S1b,c).Therefore, we co-expressed flag-tagged CNTRB 180-903aa and its PP4-binding-deficient mutant, ARVA, together with GFP, GFP-R3A, or GFP-R3B, respectively, in HEK293 cells.Using co-immunoprecipitation experiments, we demonstrated Next, we measured the frequency of HR applying the commonly used DR-GFP U2OS reporter cell line.The reporter cell line carries two truncated gfp (green fluorescent proteinencoding) cassettes: the first fragment contains an I-SceI rare cutter nuclease recognition site, and the second fragment contains the missing 466 bp to prevent GFP expression by HR action.In detail, when I-SceI cuts and generates the DSB, homologous recombination takes place using the other truncated gfp (igfp) as a template to restore the GFP cassette, resulting in functional GFP.In the end, the proportion of the GFP signal-carrying cells reflects the efficiency of the HR.To apply this approach, we detected a strong decrease in HR when we downregulated cntrob, pp4c, r3a, r3b separately, or cntrob and pp4c together (Figure 2b and Table S2).These results confirm our previous findings and suggest that PP4 cooperates with CNTRB in the regulation of HR.   (c,d) Chromosome study in CNTRB-or PP4 subunit-depleted cells.Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c.After 48 h, the cells were treated with 2 Gy X-ray irradiation and treated for 6 h with 20 µg/mL caffeine and 10 µg/mL colchicine.NT refers to non-treated (non-irradiated) cells.(c) Chromosome aberrations were counted in 150 spreads in each sample, and the percentage of arms closed phenotype was plotted.Graphs include all data points and mean ± SEM (n = 3, three replicates).Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point.(d) Representative images of chromosome spreads isolated from the indicated cells in NT or in X-ray irradiated samples.Scale bar is 10 µm.Insets at bottom left show magnified representative chromosome morphologies.
To study the effect of CNTRB, PP4c, R3A, R3B, or CNTRB and PP4 depletions in DSB repair at the chromosome level, we isolated chromosome spreads after X-ray irradiation.We observed an increased number of chromosome aberrations, particularly a phenotype characterized by closed chromosome arms.One possible explanation for this observed phenotype is that the Holliday junction structures, which form during DNA repair triggered by X-ray exposure, were inadequately resolved.As a consequence, the sister chromatids were retained instead of being separated properly.The frequency of this phenotype was significantly higher in the CNTRB-or PP4-silenced cells compared to the control group (Figure 2c,d and Table S2), suggesting that the depletion of these transcripts leads to a defect in DSB repair.Furthermore, we assessed the proportion of apoptotic cells during our examination.However, we did not observe a notably elevated fraction of apoptotic cells.As a benchmark, we evaluated H 2 O 2 -treatment, and in this case, we identified a substantial increase (Figure S2k,l).
Taken together, these results indicate that CNTRB and PP4c regulate the resolution of Holliday junction structures through cooperation in HR-mediated DNA repair.

Role of SQ and FRVP Motifs of CNTRB and Their Associated Phenotypes in the DNA Damage Response
A SILAC (stable isotope labeling with amino acids in cell culture) study has revealed that Ser781 in the SQ motif of CNTRB is phosphorylated by either ATM or ATR kinase [39].Recently, it was shown that the ATR kinase phosphorylates CNTRB, which is required for DNA repair by HR.However, the reversing phosphatase remains unknown [40,53].We note that this particular SQ motif is located adjacent to the PP4-binding site (771-FRVP-774 of CNTRB, Figure 1a), which is specifically recognized by R3A and R3B subunits of PP4 (Figure 1b,c) [29].To investigate the importance of the SQ motif (in fact, Ser781), as well as the PP4-recognition FRVP sequence in CNTRB function during DNA damage response, we generated mutated forms of CNTRB with the following amino acid substitutions: Ser781 in the SQ motif was replaced with alanine (S781A) to generate a non-phosphorylatable form, or with aspartate (S781D) to create a phosphor-mimetic form of CNTRB; and the phenylalanine (F) and proline (P) in the FRVP motif were replaced with alanine (FRVP to ARVA) to abolish the PP4 interaction (Figure 1b,c) [29].We first assessed the binding capacity of recombinant GST, GST-R3A, and GST-R3B to the 35 S-labeled full-length CNTRB-S781A or CNTRB-S781D using the GST-IVTT in vitro binding assay [43].Interestingly, none of the mutations affected the interaction (Figure 3a).We then performed an in vivo co-immunoprecipitation experiment, which showed that both GFP-R3A and GFP-R3B bound to the S781A and S781D variants of Flag-CNTRB 180-903aa (Figure 3a), similarly to the in vitro direct binding (Figure 3b).These results indicate that the interaction between CNTRB and R3A or R3B requires the intact FRVP PP4-binding motif (Figure 1b,c) but is likely independent of the SQ motif of CNTRB (Figure 3a,b).or Flag-CNTRB 180-903aa -S781D (indicated as S781D) in HEK293 cultured cells shows that CNTRB binding to R3A or R3B is independent of the integrity of the SQ motif in vivo.Cell lysate inputs and purified proteins were subjected to SDS-PAGE and western blot analysis using the indicated antibodies.Ponceau S-stained membranes are shown in Figure S4. (c,d) Chromosome study in siControl or siCNTRB cells co-transfected with empty Flag plasmid or CNTRB-depleted (siCNTRB) cells co-transfected with the wild-type or S781A/S781D/ARVA mutant variants of Flag-CNTRB 180-903aa .After 48 h, the cells were treated with 2 Gy X-ray irradiation and treated for 6 h with 20 µg/mL caffeine and 10 µg/mL colchicine.NT refers to non-treated (non-irradiated) cells.(c) Chromosome aberrations were counted in 150 spreads in each sample, and the percentage of arms closed phenotype was plotted.Graphs include all data points and mean ± SEM (n = 3, three replicates).Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point.n.s.: not significant.(d) Representative micrographs of chromosome spreads isolated from cells after X-ray irradiation or without treatment (NT).Scale bar is 10 µm.Insets at bottom left show magnified representative chromosome morphologies.
Next, we tested whether these CNTRB variants have an impact on the modulation of DNA repair progression.We co-transfected cells with Flag-tagged wild-type CNTRB 180-903aa or its ARVA, S781A or S781D derivatives, respectively, along with CNTRB siRNA.It is important to note that the siRNA we used exclusively targeted the endogenous centrob transcript (its 5 end, which is missing from the Flag-tagged mutants, Table S1), and did not affect the levels of the transgenic proteins (Figure S3).This allowed us to specifically assess the function of the indicated Flag-CNTRB derivatives.Using this experimental setup, we performed a chromosome aberration assay and observed an increased number of abnormal closed-arm morphology [5] in the siCNTRB sample upon X-ray irradiation, which was restored in cells expressing wild-type CNTRB fragment (Figure 3c,d and Table S2).However, the expression of the ARVA, S781A, or S781D mutation-containing CNTRB derivatives did not restore the function of CNTRB, suggesting that PP4 binding to CNTRB, as well as the integrity of the SQ motif, is key in the regulation of DNA repair (Figure 3c,d).
To summarize, our results emphasize the importance of PP4 binding to the single FRVP motif, as well as the ATR-targeted SQ motif, in the functioning of CNTRB, particularly in its regulation of DNA repair progression in response to DNA damage.

Discussion
Understanding the molecular etiology of cancer plays an indispensable role in shaping the future of cancer diagnosis and personalized therapy for patients.The Ser/Thr protein phosphatase 4, PP4, has been extensively studied and found to play a critical role in several cellular processes, including cell cycle regulation [15,24,30,31,[54][55][56][57][58][59][60] and DNA repair [16,17,19,[61][62][63][64].The impact of PP4 on cancer development has been investigated, revealing that alterations in PP4 expression, subcellular localization, or activity can contribute to tumorigenesis.In breast, lung, colon, and prostate cancer, an increase in PP4 expression is often observed, suggesting a possible oncogenic role of this phosphatase [65][66][67].On the other hand, a marked decrease in PP4 expression or loss of PP4 activity is observed in ovarian and cervical cancer, highlighting its tumor suppressive functions [68].Moreover, there is compelling evidence demonstrating the role of PP4 in DNA repair through its regulation and termination of signaling events [13].A specific target of PP4 is γH2AX, which becomes phosphorylated upon DNA damage.The dephosphorylation of γH2AX by PP4 is essential for restoring the normal progression of the cell cycle after the completion of DNA repair [20].
Centrosomes are multifunctional regulators of genome stability and ultimately serve as scaffolds for DNA repair proteins [69].Centrosomes are the major microtubule organizing centers in animal cells and modulate the dynamics of microtubules, too, which are required for DDR protein transportation when DNA is damaged [53,70].It has recently been shown that the daughter centriole-specific Centrobin (CNTRB), which is needed for centrosome duplication [32] and microtubule stability (regulated by the activity of NEK2 and pololike kinase 1) [36][37][38], also localizes to DSB sites and interacts with important HR factors, indicating its direct involvement in the repair process [40].CNTRB has been identified as a BRCA2-interacting protein, one of the major regulators of DNA repair by HR [32].In addition, CNTRB has been found to be phosphorylated by the ATR/ATM kinase, further supporting its role in DNA repair [39,40].We, and others, have shown that Centrobin is a genuine target of PP4 [29,30]; however, whether PP4 is involved in the DDR function of CNTRB has not yet been studied.
Through rigorous experiments, we have successfully confirmed a direct interaction between the regulatory subunits of PP4 phosphatase R3A or R3B, and full-length CNTRB (Figure 1).We provided evidence that the interaction depends on the presence of a single PP4-binding FxxP motif (FRVP) within CNTRB, with any alterations or mutations in the phenylalanine (F) and proline (P) residues potentially disrupting the binding capacity.In addition, our research has shown that knocking down CNTRB, along with members of the PP4 complex (including the catalytic subunit PP4c, and the substrate-binding subunit R3A or R3B), leads to a reduction in homologous recombination frequency (Figure 2b).This suggests that both CNTRB and the PP4 complex are actively involved in the same signaling pathway, highlighting their interconnectedness.Furthermore, our data suggest that CNTRB, as a target of the PP4 holoenzyme, has a similar influence on the status of the histone variant H2AX, which plays a crucial role in the DNA repair response (Figure 2a).Remarkably, when we selectively silenced cntrob, pp4c, r3a, r3b, or the combined knockdown of pp4c and cntrob, we observed a significant increase in a chromosome aberration called "arms closed" morphology (Figure 2c).This phenotype is likely due to incomplete Holliday junction resolution.Furthermore, CNTRB phosphorylated by ATR is required for both cell survival and homologous recombination after induction of DNA damage [40].We showed that expression of ARVA (deficient in PP4-binding), and S781A or S781D (phospho-null or phospho-mimetic forms of Ser781 in the SQ motif) variants of CNTRB could not rescue the CNTRB-silencing induced chromosome aberration phenotype in gamma-irradiated cells (Figure 3c,d) compared to wild-type CNTRB.This finding suggests that the putative phosphorylation site within the SQ motif and the PP4 consensus recognition motif, FRVP, of CNTRB are crucial in DNA repair, most probably by regulating the function of CNTRB in the resolution of the Holliday structures.Based on literature data and our results, we assume that the phosphoregulation of CNTRB might contribute to the interaction between CNTRB and BRCA2, thereby promoting the process of homologous recombination.Notably, the precise characterization of this interaction remains the focus of future research endeavors.Considering our comprehensive results, it is evident that the PP4 holoenzyme and CNTRB jointly contribute to DNA repair by homologous recombination.
In summary, our research emphasizes the interplay between PP4 and CNTRB in the context of genome instability and DNA repair.By elucidating the molecular mechanisms underlying their interactions and their roles in homologous recombination, these findings support potential therapeutic targets and treatment strategies.

Conclusions
In this research, we present supporting evidence for the physical interaction between PP4 and CNTRB.Additionally, we illustrate the essential role played by the intact FRVP and SQ motifs within CNTRB in mending double-stranded DNA breaks resulting from X-ray irradiation.These breaks are repaired through human cell homologous recombination (HR) mechanisms.When PP4 is excluded from CNTRB or mutations affect the SQ motif, an HR delay ensues.This delay leads to the accumulation of chromosomes displaying irregular morphology, likely attributable to the impaired resolution of Holliday junctions.Our discovery proposes a novel role for PP4 during the later stages of HR, further validating its participation in facilitating the resumption of the cell cycle post-DNA repair procedures.

Cells 2023 , 18 Figure 1 .
Figure 1.R3A and R3B bind directly to CNTRB through the PP4-binding motif.(a) Schematic representation of the human CNTRB protein: the PP4-binding FRVP sequence and the ATR kinaserecognition SQ motif are in proximity.Numbers indicate amino acid positions.P indicates putative (de)phosphorylation.(b) Autoradiogram (Autorad.) of GST-IVTT in vitro binding assay demonstrates that GST-R3A and GST-R3B (baits) specifically bind to 35 S-methionine-labeled CNTRB ( 35 S-CNTRB) produced in IVTT.The interaction requires an intact FRVP motif in CNTRB, because the ARVA mutant ( 35 S-CNTRB-ARVA) does not bind to the bait proteins.The Coomassie brilliant bluestained gel (CBB) represents the loading of the bait proteins.GST serves as a negative control bait.(c) GFP-trap co-immunoprecipitation of GFP (negative control), GFP-R3A or GFP-R3B transiently co-expressed with either Flag-CNTRB 180-903aa (indicated as WT) or Flag-CNTRB 180-903aa -ARVA (indicated as ARVA) in HEK293 cultured cells show that CNTRB binding to R3A or R3B requires the intact FRVP motif in vivo.Cell lysate inputs and purified proteins were subjected to SDS-PAGE and western blot analysis using the indicated antibodies.Ponceau S-stained membranes are shown in Figure S4.

Figure 1 .
Figure 1.R3A and R3B bind directly to CNTRB through the PP4-binding motif.(a) Schematic representation of the human CNTRB protein: the PP4-binding FRVP sequence and the ATR kinaserecognition SQ motif are in proximity.Numbers indicate amino acid positions.P indicates putative (de)phosphorylation.(b) Autoradiogram (Autorad.) of GST-IVTT in vitro binding assay demonstrates that GST-R3A and GST-R3B (baits) specifically bind to 35 S-methionine-labeled CNTRB ( 35 S-CNTRB) produced in IVTT.The interaction requires an intact FRVP motif in CNTRB, because the ARVA mutant ( 35 S-CNTRB-ARVA) does not bind to the bait proteins.The Coomassie brilliant blue-stained gel (CBB) represents the loading of the bait proteins.GST serves as a negative control bait.(c) GFP-trap coimmunoprecipitation of GFP (negative control), GFP-R3A or GFP-R3B transiently co-expressed with either Flag-CNTRB 180-903aa (indicated as WT) or Flag-CNTRB 180-903aa -ARVA (indicated as ARVA) in HEK293 cultured cells show that CNTRB binding to R3A or R3B requires the intact FRVP motif in vivo.Cell lysate inputs and purified proteins were subjected to SDS-PAGE and western blot analysis using the indicated antibodies.Ponceau S-stained membranes are shown in Figure S4.

Figure 2 .
Figure 2. PP4 and CNTRB act together during DNA damage response.(a) Enumeration of γH2AX foci in HeLa cells.Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c.After 48 h of RNAi, DNA damage was induced with 2 Gy X-ray irradiation and γH2AX foci were counted in 50 cells at different times (2, 4, and 8 h) post-irradiation.NT indicates the non-treated (non-irradiated) samples.Graphs include all data points and mean ± SEM (n = 3, three replicates).Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point.(b) Homologous recombination measuring assay.Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c.After 48 h, the cells were transfected with I-SceI expressing plasmid construct.The number of GFP-positive cells was calculated with Cellprofiler [52] and normalized to siControl-transfected cells.Graphs include all data points

Figure 2 .
Figure 2. PP4 and CNTRB act together during DNA damage response.(a) Enumeration of γH2AX foci in HeLa cells.Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c.After 48 h of RNAi, DNA damage was induced with 2 Gy X-ray irradiation and γH2AX foci were counted in 50 cells at different times (2, 4, and 8 h) post-irradiation.NT indicates the non-treated (non-irradiated) samples.Graphs include all data points and mean ± SEM (n = 3, three replicates).Asterisks indicate p-values (*** p < 0.001) obtained by linear regression fitted independently for each time point.(b) Homologous recombination measuring assay.Cells were transfected with the following siRNAs: siControl, siCNTRB, siPP4c, siR3A, siR3B, or co-transfected with siCNTRB and siPP4c.After 48 h, the cells were transfected with I-SceI expressing plasmid construct.The number of GFP-positive cells was calculated with Cellprofiler [52] and normalized to siControl-transfected cells.Graphs include all data points and

Figure 3 .
Figure 3.The FRVP and SQ motifs are crucial for CNTRB-regulated DNA repair.(a) Autoradiogram (autorad.) of GST-IVTT in vitro binding assay demonstrates that GST-R3A and GST-R3B (baits) interact with the S781A and S781D mutants of 35 S-methionine-labeled CNTRB ( 35 S-CNTRB-S781A and 35 S-CNTRB-S781D) produced in IVTT.The Coomassie brilliant blue-stained gel (CBB) represents the loading of the bait proteins.GST serves as a negative control bait.(b) GFP-trap co-immunoprecipitation of GFP (negative control), GFP-R3A or GFP-R3B transiently co-expressed with either Flag-CNTRB180-903aa (indicated as WT), Flag-CNTRB 180-903aa -S781A (indicated as S781A), or Flag-CNTRB 180- 903aa -S781D (indicated as S781D) in HEK293 cultured cells shows that CNTRB binding to R3A or R3B is independent of the integrity of the SQ motif in vivo.Cell lysate inputs and purified proteins were subjected to SDS-PAGE and western blot analysis using the indicated antibodies.Ponceau S-stained