Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.

Phosphatase 1 nuclear targeting subunit is an essential regulator of M-phase entry, maintenance, and exit.

Mitotic progression is regulated largely through dynamic and reversible protein phosphorylation that is modulated by opposing actions of protein kinases and phosphatases. In this study, we show that phosphatase 1 nuclear targeting subunit (Pnuts) functions as a master regulator of mitosis by modulating protein phosphatase 1 (PP1). Overexpression of Pnuts in Xenopus egg extracts inhibited both mitotic and meiotic exit. Immunodepletion of Pnuts from egg extracts revealed its essential functions in mitotic entry and maintenance. The level of Pnuts oscillates during the cell cycle and peaks in mitosis. Pnuts destruction during M-phase exit is mediated by the anaphase-promoting complex/cyclosome (APC/C)-targeted ubiquitination and proteolysis, and conserved destruction motifs of Pnuts. Disruption of Pnuts degradation delayed M-phase exit, suggesting it as an important mechanism to permit M-phase exit.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing CDKs. However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

Greatwall and Polo-like kinase 1 coordinate to promote checkpoint recovery.

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

The MASTL-ENSA-PP2A/B55 axis modulates cisplatin resistance in oral squamous cell carcinoma.

Platinum-based chemotherapy is the standard first-line treatment for oral squamous cell carcinoma (OSCC) that is inoperable, recurrent, or metastatic. Platinum sensitivity is a major determinant of patient survival in advanced OSCC. Here, we investigated the involvement of MASTL, a cell cycle kinase that mediates ENSA/ARPP19 phosphorylation and PP2A/B55 inhibition, in OSCC therapy. Interestingly, upregulation of MASTL and ENSA/ARPP19, and downregulation of PP2A/B55, were common in OSCC. MASTL expression was in association with poor patient survival. In established OSCC cell lines, upregulation of MASTL and ENSA, and downregulation of B55 genes, correlated with cisplatin resistance. We further confirmed that stable expression of MASTL in OSCC cells promoted cell survival and proliferation under cisplatin treatment, in an ENSA-dependent manner. Conversely, deletion of MASTL or ENSA, or overexpression of B55α, sensitized cisplatin response, consistent with increased DNA damage accumulation, signaling, and caspase activation. Moreover, GKI-1, the first-in-class small molecule inhibitor of MASTL kinase, phenocopied MASTL depletion in enhancing the outcome of cisplatin treatment in OSCC cells, at a dose substantially lower than that needed to disrupt mitotic entry. Finally, GKI-1 exhibited promising efficacy in a mouse tumor xenograft model, in conjunction with cisplatin therapy.

PARP1 Upregulation in Recurrent Oral Cancer and Treatment Resistance.

First-line treatments for oral cancer typically include surgery, radiation, and in some cases, chemotherapy. Radiation and oral cancer chemotherapeutics confer cytotoxicity largely by inducing DNA damage, underscoring the importance of the cellular DNA damage repair and response pathways in cancer therapy. However, tumor recurrence and acquired resistance, following the initial response to treatment, remains as a major clinical challenge. By analyzing oral tumor cells derived from the primary and recurrent tumors of the same patient, our study revealed upregulated PARP1 expression in the recurrent tumor cells. Cisplatin and 5-fluorouracil treatment further augmented PARP1 expression in the recurrent, but not the primary, tumor cells. Post-treatment upregulation of PARP1 was dependent on the catalytic activities of PARP and CDK7. Consistent with the established function of PARP1 in DNA repair, we showed that overexpression of PARP1 rendered the primary tumor cells highly resistant to DNA damage treatment. Conversely, PARP inhibition partially reversed the treatment resistance in the recurrent tumor cells; combinatorial treatment using a PARP inhibitor and cisplatin/5-fluorouracil significantly sensitized the tumor response in vivo. Taken together, we reported here PARP1 upregulation as a clinically relevant mechanism involved in oral cancer recurrence, and suggested the clinical benefit of PARP inhibitors, currently approved for the treatment of several other types of cancer, in oral cancer.

The Sm core components of small nuclear ribonucleoproteins promote homologous recombination repair.

DNA Double strand breaks (DSBs) are highly hazardous to the cell, and are repaired predominantly via non-homologous end joining (NHEJ) and homologous recombination (HR). Using DSB-mimicking DNA templates, our proteomic studies identified a group of Sm core proteins of small nuclear ribonucleoproteins (snRNPs) as potential DSB-associated proteins. We further confirmed that these Sm proteins were recruited to laser-induced DNA damage sites, and co-localized with established DNA damage repair factors. Depletion of Sm-D3 or Sm-B induced accumulation of γ-H2AX, and impaired the repair efficiency of HR, but not NHEJ. Furthermore, disruption of Sm-D3 reduced the protein level of HR factors, especially RAD51 and CHK1, but caused no change in the expression of repair factors involved in NHEJ. Mechanistically, Sm-D3 proteins bound RAD51, suppressed the ubiquitination of RAD51, and mediated the stabilization of RAD51; Sm-D3 depletion particularly impacted the level of RAD51 and CHK1 on damaged chromatin. As such, our studies characterized a role of Sm proteins in HR repair, via a new mechanism that is distinct from their conventional functions in RNA processing and gene regulation, but consistent with their direct recruitment to DNA damage sites and association with repair factors.

Discovery of Halogenated Benzothiadiazine Derivatives with Anticancer Activity*.

Mitochondrial respiratory complex II (CII), also known as succinate dehydrogenase, plays a critical role in mitochondrial metabolism. Known but low potency CII inhibitors are selectively cytotoxic to cancer cells including the benzothiadiazine-based anti-hypoglycemic diazoxide. Herein, we study the structure-activity relationship of benzothiadiazine derivatives for CII inhibition and their effect on cancer cells for the first time. A 15-fold increase in CII inhibition was achieved over diazoxide, albeit with micromolar IC50 values. Cytotoxicity evaluation of the novel derivatives resulted in the identification of compounds with much greater antineoplastic effect than diazoxide, the most potent of which possesses an IC50 of 2.93±0.07 µM in a cellular model of triple-negative breast cancer, with high selectivity over nonmalignant cells and more than double the potency of the clinical agent 5-fluorouracil. No correlation between cytotoxicity and CII inhibition was found, thus indicating an as-yet-undefined mechanism of action of this scaffold. The derivatives described herein represent valuable hit compounds for therapeutic discovery in triple-negative breast cancer.

Undamaged DNA transmits and enhances DNA damage checkpoint signals in early embryos.

In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require gamma-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

Kinesin Kif2C in regulation of DNA double strand break dynamics and repair.

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

DNA can be damaged in many ways, and a double strand break is one of the most dangerous. This occurs when both strands of the double helix snap at the same time, leaving two broken ends. When cells detect this kind of damage, they race to get it fixed as quickly as possible. Fixing these double strand breaks is thought to involve the broken ends being moved to 'repair centers' in the nucleus of the cell, but it was unclear how the broken ends were moved. One possibility was that the cells transport the broken ends along protein filaments called microtubules. Cells can assemble these track-like filaments on-demand to carry cargo attached to molecular motors called kinesins. However, this type of transport happens outside of the cell's nucleus, and while there are different kinesin proteins localized inside the nucleus, their roles are largely unknown. In an effort to understand how broken DNA ends are repaired, Zhu, Paydar et al. conducted experiments that simulated double strand breaks and examined the proteins that responded. The first set of experiments involved mixing cut pieces of DNA with extracts taken from frog eggs or human cells. Zhu, Paydar et al. found that one kinesin called Kif2C stuck to the DNA fragments, and attached to many proteins known to play a role in DNA damage repair. Kif2C had previously been shown to help separate the chromosomes during cell division. To find out more about its potential role in DNA repair, Zhu, Paydar et al. then used a laser to create breaks in the DNA of living human cells and tracked Kif2C movement. The kinesin arrived within 60 seconds of the DNA damage and appeared to transport the cut DNA ends to 'repair centers'. Getting rid of Kif2C, or blocking its activity, had dire effects on the cells' abilities to mobilize and repair breaks to its DNA. Without the molecular motor, fewer double strand breaks were repaired, and so DNA damage started to build up. Defects in double strand break repair happen in many human diseases, including cancer. Many cancer treatments damage the DNA of cancer cells, sometimes in combination with drugs that stop cells from building and using their microtubule transport systems. Understanding the new role of Kif2C in DNA damage repair could therefore help optimize these treatment combinations.

Phosphatase 1 Nuclear Targeting Subunit (PNUTS) Regulates Aurora Kinases and Mitotic Progression.

Mitotic progression is regulated largely by reversible phosphorylation events that are mediated by mitotic kinases and phosphatases. Protein phosphatase 1 (PP1) has been shown to play a crucial role in regulation of mitotic entry, progression, and exit. We previously observed, in Xenopus egg extracts, that phosphatase 1 nuclear targeting subunit (PPP1R10/PNUTS) acts as a mitotic regulator by negatively modulating PP1. This study investigates the role of PNUTS in mitotic progression in mammalian cells, and demonstrates that PNUTS expression is elevated in mitosis and depletion partially blocks mitotic entry. Cells that enter mitosis after PNUTS knockdown exhibit frequent chromosome mis-segregation. Aurora A/B kinase complexes and several kinetochore components are identified as PNUTS-associated proteins. PNUTS depletion suppresses the activation of Aurora A/B kinases, and disrupts the spatiotemporal regulation of the chromosomal passenger complex (CPC). PNUTS dynamically localizes to kinetochores, and is required for the activation of the spindle assembly checkpoint. Finally, PNUTS depletion sensitizes the tumor cell response to Aurora inhibition, suggesting that PNUTS is a potential drug target in combination anticancer therapy. IMPLICATIONS: Delineation of how PNUTS governs the mitotic activation and function of Aurora kinases will improve the understanding of the complex phospho-regulation in mitotic progression, and suggest new options to enhance the therapeutic efficacy of Aurora inhibitors.

[Modified Ilizarov hip reconstruction in treatment of adolescent hip instability].

OBJECTIVE: To evaluate the effectiveness of modified Ilizarov hip reconstruction in the treatment of hip instability. METHODS: The clinical data of 13 young patients with hip diseases treated with modified Ilizarov hip reconstruction between January 2010 and March 2018 were retrospectively analyzed. There were 2 males and 11 females, aged from 14 to 34 years, with an average age of 24.2 years. There were 1 case of hip dysplasia and dislocation due to spinal bifida, 3 cases of hip dysplasia after pyogenic arthritis of the hip, 2 cases of developmental dysplasiaof the hip (DDH) accompanying femoral head necrosis who rejected hip replacement, 6 cases of young DDH refused to undergo hip replacement, and 1 case of bilateral hip dysplasia with dislocation due to sputum cerebral palsy. The disease duration was 2-20 years, with an average of 8.5 years. Preoperative Trendelenburg sign was positive in 12 cases and negative in 1 case. The preoperative Harris score of hip joint was 53.5±8.9 and the unequal length of lower limbs was (46.08±15.73) mm. Postoperative Harris hip score and patients' satisfaction with effectiveness evaluated according to their self scoring were used to assess the effectiveness. RESULTS: All 13 patients were followed up 1-5 years, with an average of 2.6 years. Five patients developed postoperative needle infection, which improved after dressing change; 7 patients had limited knee joint activity and improved after knee joint function training. The Trendelenburg sign was negative at 1 year after operation, and the patient's hip pain symptoms were relieved or disappeared. The Harris hip score of patients at 1 year after operation was 84.5±6.1, which was significantly improved when compared with preoperative one ( t=－10.538, P=0.000). According to Harris hip score, the effectiveness results were excellent in 4 cases, good in 5 cases, and fair in 4 cases, with an excellent and good rate of 69.2%. The unequal length of lower limbs was (15.38±7.27) mm, which was significantly better than that before operation ( t=11.826, P=0.000). At last follow-up, the patients' satisfaction score was 80%-95%, with an average of 88%. CONCLUSION: Modified Ilizarov hip reconstruction can be used to treat young patients with hip disease who are unsuitable or refuse to undergo artificial hip replacement. Its effectiveness is reliable, and it has unique advantages in limb limp improvement and limb shortening correction.

[Minimally invasive external fixation of humeral supracondylar angular rotation center for the treatment of cubitus varus deformity in adolescents].

OBJECTIVE: To investigate clinical effect of minimally invasive osteotomy and external fixation with the center of roration of angulation (CORA) in treating cubitus varus in adolescents. METHODS: From August 2013 to August 2017, 15 patients with cubitus varus caused by supracondylar fracture of humerus were treated with minimally invasive osteotomy and external fixation with the CORA. Among them, including 9 males and 6 females; 11 patients on the left side and 4 patients on the right side; aged from 13 to 16 years old with an average of 14.5 years old. The time from injury to operation was for 6 to 10 years with an average of 7.5 years. Five patients had a history of recurrence after cubitus varus surgery. Correction time. fracture healing time, carrying angle were observed, Laupattarakasem standard was used to evaluate clinical effect. RESULTS: All patients were followed up from 12 to 30 months with an average of 24 months; correction time ranged from 3 to 5 weeks with an average of 4 weeks; fracture healing time ranged from 4 to 6 months with an average of 5 months; carrying angle before operation ranged from -12° to -23°, and improved 9° to 14° after operation. According to Laupattarakasem evaluation criteria, 11 patients got an excellent result, 3 good and 1 fair. CONCLUSIONS: Minimally invasive osteotomy and external fixation with CORA in treating cubitus varus deformity in adolescents has advantages of less trauma, less blood loss, earlier exercise, speed and angle of correction could controlled without hospitalized for fixation.

Phosphatase 1 Nuclear Targeting Subunit Mediates Recruitment and Function of Poly (ADP-Ribose) Polymerase 1 in DNA Repair.

PARP, particularly PARP1, plays an essential role in the detection and repair of DNA single-strand breaks and double-strand breaks. PARP1 accumulates at DNA damage sites within seconds after DNA damage to catalyze the massive induction of substrate protein poly ADP-ribosylation (PARylation). However, the molecular mechanisms underlying the recruitment and activation of PARP1 in DNA repair are not fully understood. Here we show that phosphatase 1 nuclear targeting subunit 1 (PNUTS) is a robust binding partner of PARP1. Inhibition of PNUTS led to strong accumulation of endogenous DNA damage and sensitized the cellular response to a wide range of DNA-damaging agents, implicating PNUTS as an essential and multifaceted regulator of DNA repair. Recruitment of PNUTS to laser-induced DNA damage was similar to that of PARP1, and depletion or inhibition of PARP1 abrogated recruitment of PNUTS to sites of DNA damage. Conversely, PNUTS was required for efficient induction of substrate PARylation after DNA damage. PNUTS bound the BRCA1 C-terminal (BRCT) domain of PARP1 and was required for the recruitment of PARP1 to sites of DNA damage. Finally, depletion of PNUTS rendered cancer cells hypersensitive to PARP inhibition. Taken together, our study characterizes PNUTS as an essential partner of PARP1 in DNA repair and a potential drug target in cancer therapy. SIGNIFICANCE: These findings reveal PNUTS as an essential functional partner of PARP1 in DNA repair and suggest its inhibition as a potential therapeutic strategy in conjunction with DNA-damaging agents or PARP inhibitors.See related commentary by Murai and Pommier, p. 2460.

[Effectiveness of minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening].

Objective: To explore the effectiveness of minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening. Methods: Seventy-one patients with femoral shortening deformity who met the selection criteria between January 2013 and June 2016 were randomly divided into trial group (36 cases were treated with minimally invasive osteotomy Ilizarov technique combined with intramedullary nail for femoral lengthening) and control group (35 cases were treated with simple Ilizarov technique for femoral lengthening). There was no significant difference in age, gender, causes of femoral shortening, length of femoral shortening, rate of femoral deformity between the two groups ( P>0.05). The operation duration, intraoperative blood loss, lengthening rate, external fixation duration, frequency of pin tract infection, osteotomy healing time, and range of motion (ROM) of knee at 1 year after operation were recorded and compared between the two groups. Results: The patients of two groups were followed up 12-60 months (mean, 31 months). Pin tract infection occured in 8 cases (10 pins), including 1 case (1 pin) in the trial group and 7 cases (9 pins) in the control group. There was significant difference in the incidence of pin tract infection between the two groups ( χ2=5.265, P=0.022). All patients were cured by replacing the fixation pins, changing dressing actively, application of antibiotics, and adequate postoperative care. The operation duration, intraoperative blood loss, external fixation duration, osteotomy healing time, and ROM of knee at 1 year after operation of the trial group were superior to those of the control group, showing significant differences ( P<0.05). There was no significant difference in the lengthening rate between the two groups ( t=-1.581, P=0.153). Conclusion: The minimally invasive osteotomy Ilizarov technique combined with intramedullary nail in femoral lengthening increases the operation time, but the external fixation duration and incidence of pin tract infection are significantly reduced and the function of knee is significantly improved.

Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress.

The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55ß in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55ß-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55ß, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.

NFBD1/Mdc1 mediates ATR-dependent DNA damage response.

Budding yeast Rad9 (scRad9) plays a central role in mediating Mec1-dependent phosphorylation by recruiting its downstream substrates. The human scRad9 orthologues 53BP1 and NFBD1 associate with ionizing radiation-induced foci (IRIF) at sites of DNA repair. RNAi-based gene silencing of 53BP1 or NFBD1 has shown impaired phosphorylation of SQ/TQ [ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) substrates] at IRIF, intra-S, and G(2)-M checkpoints and has thereby revealed essential roles for 53BP1 and NFBD1 in the DNA damage signaling pathway. Whether 53BP1 and NFBD1 are required for activation of kinases and/or for recruitment of substrates at IRIF, however, is not clear. Here we show that both 53BP1 and NFBD1 are required for recruitment of ATR to DNA damage sites, as well as for ATR-dependent phosphorylation in response to DNA damage. NFBD1 is not required for ssDNA generation at DNA damage sites and is not recruited by replication protein A (RPA)-coated ssDNA. We therefore show that recruitment of NFBD1 and/or 53BP1, the factors downstream of H2AX, is independent of ssDNA generation and RPA coating, whereas both ssDNA and RPA coating play key roles in regulation of the ATR-dependent pathway. These novel findings help clarify where NFBD1 functions in DNA damage early responses.