DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

Association Between Commuting Time and Subjective Well-Being in Relation to Regional Differences in Korea.

BACKGROUND: Long commuting times have a negative impact on mental health. However, few studies have explored the relationship between commuting time and well-being based on urbanization by region. Our study examines this relationship as well as the effect of regional differences on Korean workers. METHODS: We used data from the sixth Korean Working Conditions Survey. Commuting time and occupational factors were assessed using a questionnaire, and subjective well-being was assessed using the World Health Organization-5 Well-Being Index. Regions were divided into the cities and the provinces based on Korea's administrative divisions. Logistic regression analysis was performed to investigate the association between commuting time and well-being. Adjusted odds ratios (aORs) with 95% confidence intervals (CIs) for well-being were estimated, using participants commuting time of < 20 minutes as a reference group. RESULTS: The total number of workers was 29,458 (13,855 men, 15,603 women). We found higher aORs for low well-being among workers with long commuting times (aOR, 1.23; 95% CI, 1.11-1.36 and aOR, 1.28; 95% CI, 1.16-1.42 for 60-79 and ≥ 80 minutes, respectively). When stratified by sex and region, higher aORs for low well-being were found only in the workers who lived in cities. CONCLUSION: Long commuting time was negatively associated with well-being in Korean wage workers living in the cities. Policies for reducing commuting time should be discussed to address the mental health of workers, especially those living in metropolitan cities.

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase.

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku's affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.

Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments.

Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability.

Exposure assessment of particulate matter and blood chromium levels in people living near a cement plant.

This study evaluates the effect of air pollution caused by cement plants on nearby residential areas and performs an exposure assessment of particulate matter (PM) and total Cr, Cr6+, Pb, and Al. Further, the blood Cr levels of residents exposed to PM released by cement plants are also assessed. Nine buildings (eight residential and one elementary school building) close to cement plants were selected for this study, which were located in Pyeongtaek port, in west of Gyeonggi Province, South Korea. A total of 51 suspended particulate samples were collected at a flow rate of 2.0 L/min. Total Cr was more widely detected in residents' houses and elementary schools. PM levels were higher at distances of 4.1 and 4.8 km than those at closer distances of 2.7 km. This was due to the influence of wind direction. The estimated mean blood level of Cr for the study participants was 3.80 µg/L, which is higher than levels estimated by other studies on Cr blood levels. Therefore, cement plants could cause an increase in total Cr and blood Cr levels in residential areas, and more continuous monitoring is necessary to better understand their impacts.

DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.

BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle.

Non-homologous end-joining (NHEJ) and homologous recombination (HR) are the two prominent pathways responsible for the repair of DNA double-strand breaks (DSBs). NHEJ is not restricted to a cell-cycle stage, whereas HR is active primarily in the S/G2 phases suggesting there are cell cycle-specific mechanisms that play a role in the choice between NHEJ and HR. Here we show NHEJ is attenuated in S phase via modulation of the autophosphorylation status of the NHEJ factor DNA-PKcs at serine 2056 by the pro-HR factor BRCA1. BRCA1 interacts with DNA-PKcs in a cell cycle-regulated manner and this interaction is mediated by the tandem BRCT domain of BRCA1, but surprisingly in a phospho-independent manner. BRCA1 attenuates DNA-PKcs autophosphorylation via directly blocking the ability of DNA-PKcs to autophosphorylate. Subsequently, blocking autophosphorylation of DNA-PKcs at the serine 2056 phosphorylation cluster promotes HR-required DNA end processing and loading of HR factors to DSBs and is a possible mechanism by which BRCA1 promotes HR.

The N-terminal region of the DNA-dependent protein kinase catalytic subunit is required for its DNA double-stranded break-mediated activation.

DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1-2713), termed N-PKcs, and the C terminus (amino acids 2714-4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity.

Phosphorylation of the cryptochrome 1 C-terminal tail regulates circadian period length.

The Cryptochrome (CRY) proteins are critical components of the mammalian circadian clock and act to rhythmically repress the activity of the transcriptional activators CLOCK and BMAL1 at the heart of the clock mechanism. The CRY proteins are part of a large repressive complex, the components of which are not completely known. Using mass spectroscopy, we identified the catalytic subunit of DNA-dependent protein kinase as a CRY-interacting protein and found that loss or inhibition of this kinase results in circadian rhythms with abnormally long periods. We then identified serine 588 in the C-terminal tail of mouse CRY1 as a potential DNA-PK phosphorylation site but surprisingly found that the phosphomimetic mutation S588D also results in long period rhythms, similar to the loss of DNA-PK. Consistent with this, we found that phosphorylation of this site is increased in cells lacking DNA-PK, suggesting that DNA-PK negatively regulates the phosphorylation of this site most likely through indirect means. Furthermore, we found that phosphorylation of this site increases the stability of the CRY1 protein and prevents FBXL3-mediated degradation. The phosphorylation of this site is robustly rhythmic in mouse liver nuclei, peaking in the middle of the circadian day at a time when CRY1 levels are declining. Therefore, these data suggest a new role for the C-terminal tail of CRY1 in which phosphorylation rhythmically regulates CRY1 stability and contributes to the proper circadian period length.

Clinical value of endobronchial ultrasound findings for predicting nodal metastasis in patients with suspected lymphadenopathy: a prospective study.

We evaluated whether sonographic findings can provide additional diagnostic yield in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), and can more accurately predict nodal metastasis than chest computed tomography (CT) or positron emission tomography (PET)/CT scans. EBUS-TBNA was performed in 146 prospectively recruited patients with suspected thoracic lymph node involvement on chest CT and PET/CT from June 2012 to January 2013. Diagnostic yields of EBUS finding categories as a prediction model for metastasis were evaluated and compared with findings of chest CT, PET/CT, and EBUS-TBNA. In total, 172 lymph nodes were included in the analysis: of them, 120 were malignant and 52 were benign. The following four EBUS findings were predictive of metastasis: nodal size ≥10 mm, round shape, heterogeneous echogenicity, and absence of central hilar structure. A single EBUS finding did not have sufficient diagnostic yield; however, when the lymph node had any one of the predictive factors on EBUS, the diagnostic yields for metastasis were higher than for chest CT and PET/CT, with a sensitivity of 99.1% and negative predictive value of 83.3%. When any one of predictive factors is observed on EBUS, subsequent TBNA should be considered, which may provide a higher diagnostic yield than chest CT or PET/CT.

Association between commuting time and work-related low back pain with respect to sports and leisure activities in Korean workers.

Long commuting times can induce work-related low back pain (LBP), which can be exacerbated by reduced sports and leisure activities. However, there is a lack of empirical research on commuting time and work-related LBP in Korea. In this study, we aimed to investigate the relationship between commuting time and work-related LBP as well as the effect of sports and leisure activities on Korean workers. We utilized data from the sixth Korean Working Conditions Survey to analyze the relationship between commuting time and work-related LBP using multivariable logistic regression. The total number of included workers was 28,202. Workers without sports and leisure activities, and long commuting times (40-59, 60-79, and ≥80 min) showed significantly higher odds ratios for work-related LBP (1.29 [95% Confidence intervals=1.12-1.49], 1.42 [1.22-1.65], and 1.96 [1.68-2.28], respectively). However, in workers with sports and leisure activities, the results were significant only for commuting times of 60-79 and ≥80 min (1.41 [1.13-1.75], 1.60 [1.28-1.99], respectively). Long commuting times were associated with work-related LBP, and engagement in sports and leisure activities was found to play a role in mitigating the impact among Korean wage workers.

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex.

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.

Combined effect of work from home and work during nonwork time on sleep disturbance.

Background: Owing to the coronavirus disease 2019 pandemic, being exposed to work from home and work during nonwork time simultaneously can lead to sleep disturbance; however, their combined effect is unclear. We aimed to investigate the combined effect of work from home and work during nonwork time on sleep disturbance. Methods: This study used data from the Sixth Korean Working Condition Survey and included 27,473 paid workers. Logistic regression analysis was conducted to investigate the relationship between work from home, work during nonwork time, and sleep disturbance according to sex. We re-classified participants into 4 groups based on their working from home (No/Yes) and working during nonwork time (No/Yes). The relative excess risk due to interaction was calculated to examine the effect of exposure to both telecommuting and non-regular work hours on sleep disturbance. Results: Workers exposed to work from home and work during nonwork time had significantly higher risks of sleep disturbance for all, men, and women workers (OR [95% CI]: 1.71 [1.46-2.02], 1.79 [1.43-2.23], and 1.64 [1.29-2.08] for work from home and 3.04 [2.70-3.42], 3.61 [3.09-4.22], and 2.41 [2.01-2.90] for work during nonwork time, respectively). Compared to those who were not exposed to both factors, when workers had both job factors, the ORs (95% CI) of sleep disturbance for all, men, and women were 3.93 (2.80-5.53), 5.08 (3.21-8.03), and 2.91 (1.74-4.87), respectively. The relative excess risk due to interaction of work from home and work during nonwork time was not significant for sleep disturbance. Conclusions: Work from home and work during nonwork time were each associated with sleep disturbance, but the interaction between the two factors on sleep disturbance was not observed in both men and women.

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non-homologous end joining pathway. When DSBs occur, DNA-PKcs is rapidly phosphorylated at both the Thr-2609 and Ser-2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA-PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA-PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demonstrate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation.

Perfluorooctanoic acid exposure is associated with elevated homocysteine and hypertension in US adults.

OBJECTIVE: To investigate the association between serum perfluorooctanoic acid (PFOA) concentration and cardiovascular disease, as measured by homocysteine level and blood pressure in a representative sample of US adults. METHODS: A cross-sectional study of 2934 adults (≥20 years) who participated in the 2003-2004 and 2005-2006 National Health and Nutrition Examination Survey and had detectable levels of PFOA in their serum. The health effects analysed as potentially associated with PFOA exposure included homocysteine level and blood pressure. RESULTS: The geometric mean value (95% CI) of the study participants' serum PFOA concentration was 4.00 µg/l (95% CI 3.86 to 4.13). The homocysteine and systolic blood pressure were shown to increase significantly with an increase in the log-transformed serum PFOA concentration, after adjusting for potential confounding variables. Adjusted ORs comparing participants at the 80th versus the 20th percentiles were 2.62 for hypertension (95% CI 2.09 to 3.14), and a positive association was also evident in models based on quartiles or based on restricted cubic splines. CONCLUSION: These findings suggest that background exposure to PFOA may continue a risk factor for the development of cardiovascular diseases.

Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

Progression rate from new-onset pre-hypertension to hypertension in Korean adults.

BACKGROUND: There are limited studies conducted in Asia to investigate the progression rate to hypertension (HTN). This study was done to estimate the progression rate of new-onset pre-HTN (PreHTN) to HTN during an 8-year follow-up period, and to compare the impact of PreHTN on progression to HTN. METHODS AND RESULTS: A total of 49,228 participants, aged 30 to 54 years with new-onset PreHTN at baseline (1994-1996) from a biennial national medical exam were enrolled and followed up every 2 years until 2004. The incidence rate recorded at each interval and the cumulative incidence rate of HTN were analyzed. Hazard ratio of high-normal and high blood pressure (BP) in men and women was calculated. The cumulative incidence rate for high-normal BP was 27.6% and 26.4% at 2-year follow-up, increased to respectively 64.1% and 55.8% in men and women at the 8-year follow-up. Compared to optimal BP, hazard ratios for men with high-normal BP across all age groups were 3- to 4-fold higher at 2-year, and 2- to 3-fold higher at 8-year follow-up. Hazard ratios for women were about 6-fold higher at 2-year and around 4-fold higher at 8-year follow-up. CONCLUSIONS: New PreHTN was a significant predisposing factor for future HTN, in young adults and the effect is more prominent in women.

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

Association between split shift work and work-related injury and disease absence.

BACKGROUND: Shift work (particularly split shifts) has been noted among the working conditions that hinder sustainable work. However, little is known regarding the effects of split shifts on health. This study aimed to investigate the association between split shift work and work-related injury and disease absence. METHODS: This study used data from the fifth Korean Working Conditions Survey and included 4,042 paid shift workers. Shift work type and self-reported work-related injury and disease absence were investigated using a questionnaire. Logistic regression analysis was performed to investigate the association between split shift work and work-related absence with rotating shift worker as the reference group. RESULTS: Of the 4,042 shift workers, 980 (24.3%) were split shift workers. The adjusted odds ratio (aOR) of split shift for absence due to work-related injury was high at 2.94 (95% confidence interval [CI]: 1.85-4.68) and the aOR of split shift for absence due to work-related disease was also high at 1.58 (95% CI: 1.09-2.29) compared to rotating shift. CONCLUSIONS: Split shift work leads to an increased risk of absences due to work-related injury and disease.

Effects of work organization on the occurrence and resolution of sleep disturbances among night shift workers: a longitudinal observational study.

This study aimed to investigate the association between work organization and the trajectories of insomnia patterns among night shift workers in a hospital. The health examination data of hospital workers, recorded from January 2014 to December 2018, were collected; 6765 records of 2615 night shift workers were included. Insomnia was defined as a score of ≥ 15 on the Insomnia Severity Index (ISI). Participants were categorized into five groups according to insomnia patterns derived from the analysis of their ISI scores. Work organization and socio-demographic characteristics were also investigated. Generalized estimating equation models and linear mixed models were constructed to analyze the longitudinal data. Of the total participants, 53.0% reported insomnia at least once during the follow-up period. The lack of nap opportunities and work-time control was associated with the occurrence of insomnia, whereas more than 5 years of shift work experience was related to the resolution of insomnia. All work-related factors were significantly related to insomnia risk; however, the effects were not significant in the sustained insomnia group. Although sleep problems are inevitable in night shift workers, well-designed work schedules and better work organization can help reduce the occurrence of insomnia among them.