Nonhomologous end-joining (NHEJ) is a major DNA double-strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin ...opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate-specific NHEJ factor that can be autophosphorylated or transphosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated transphosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strict autophosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcsKD/KD mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations.
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•DNA-PKcs protein inhibits direct end-ligation at the DNA ends•Intermolecular autophosphorylation of DNA-PKcs is required for end-ligation•DNA-PKcs protein, but not its kinase activity, is required for end-processing•ATM phosphorylates DNA-PKcs to recruit Artemis and promote end-processing
Differential phosphorylation of DNA-PKcs at DNA ends is the molecular switch between end-processing and end-ligation. ATM-mediated phosphorylation of DNA-PKcs is necessary for Artemis recruitment and end-processing, while intermolecular autophosphorylation of DNA-PKcs relieves the physical block on end-ligation imposed by the DNA-PKcs protein at DNA ends and is required for direct end-ligation.
Introduction:
Diabetes has emerged as an important risk factor for severe illness and death from COVID-19. There is a paucity of information on glycemic control among hospitalized COVID-19 patients ...with diabetes and acute hyperglycemia.
Methods:
This retrospective observational study of laboratory-confirmed COVID-19 adults evaluated glycemic and clinical outcomes in patients with and without diabetes and/or acutely uncontrolled hyperglycemia hospitalized March 1 to April 6, 2020. Diabetes was defined as A1C ≥6.5%. Uncontrolled hyperglycemia was defined as ≥2 blood glucoses (BGs) > 180 mg/dL within any 24-hour period. Data were abstracted from Glytec’s data warehouse.
Results:
Among 1122 patients in 88 U.S. hospitals, 451 patients with diabetes and/or uncontrolled hyperglycemia spent 37.8% of patient days having a mean BG > 180 mg/dL. Among 570 patients who died or were discharged, the mortality rate was 28.8% in 184 diabetes and/or uncontrolled hyperglycemia patients, compared with 6.2% of 386 patients without diabetes or hyperglycemia (P < .001). Among the 184 patients with diabetes and/or hyperglycemia who died or were discharged, 40 of 96 uncontrolled hyperglycemia patients (41.7%) died compared with 13 of 88 patients with diabetes (14.8%, P < .001). Among 493 discharged survivors, median length of stay (LOS) was longer in 184 patients with diabetes and/or uncontrolled hyperglycemia compared with 386 patients without diabetes or hyperglycemia (5.7 vs 4.3 days, P < .001).
Conclusion:
Among hospitalized patients with COVID-19, diabetes and/or uncontrolled hyperglycemia occurred frequently. These COVID-19 patients with diabetes and/or uncontrolled hyperglycemia had a longer LOS and markedly higher mortality than patients without diabetes or uncontrolled hyperglycemia. Patients with uncontrolled hyperglycemia had a particularly high mortality rate. We recommend health systems which ensure that inpatient hyperglycemia is safely and effectively treated.
The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells ...undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A
mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A
B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology—all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.
The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor
. KU binds to DNA ends, ...initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ
. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma
. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA
. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.
Climate change is increasing heat exposure in places such as Central America, a tropical region with generally hot/humid conditions. Working people are at particular risk of heat stress because of ...the intrabody heat production caused by physical labor. This article aims to describe the risks of occupational heat exposure on health and productivity in Central America, and to make tentative estimates of the impact of ongoing climate change on these risks. A review of relevant literature and estimation of the heat exposure variable wet bulb globe temperature (WBGT) in different locations within the region were used to estimate the effects. We found that heat stress at work is a real threat. Literature from Central America and heat exposure estimates show that some workers are already at risk under current conditions. These conditions will likely worsen with climate change, demonstrating the need to create solutions that will protect worker health and productivity.
The First International Research Workshop on Mesoamerican Nephropathy (MeN) met in Costa Rica in November 2012 to discuss how to establish the extent and degree of MeN, examine relevant causal ...hypotheses, and focus efforts to control or eliminate the disease burden. MeN describes a devastating epidemic of chronic kidney disease of unknown origin predominantly observed among young male sugarcane cutters. The cause of MeN remains uncertain; however, the strongest hypothesis pursued to date is repeated episodes of occupational heat stress and water and solute loss, probably in combination with other potential risk factor(s), such as nonsteroidal anti-inflammatory drug and other nephrotoxic medication use, inorganic arsenic, leptospirosis, or pesticides. At the research workshop, clinical and epidemiologic case definitions were proposed in order to facilitate both public health and research efforts. Recommendations emanating from the workshop included measuring workload, heat, and water and solute loss among workers; quantifying nephrotoxic agents in drinking water and food; using biomarkers of early kidney injury to explore potential causes of MeN; and characterizing social and working conditions together with methods for valid data collection of exposures and personal risk factors. Advantages and disadvantages of different population study designs were detailed. To elucidate the etiology of MeN, multicountry studies with prospective cohort design, preferably integrating an ecosystem health approach, were considered the most promising. In addition, genetic, experimental, and mechanistic methods and designs were addressed, specifically the need for kidney biopsy analysis, studies in animal models, advances in biomarkers, genetic and epigenetic studies, a common registry and repository of biological and demographic data and/or specimens, and other areas of potential chronic kidney disease experimental research. Finally, in order to improve international collaboration on MeN, workshop participants agreed to establish a research consortium to link these Mesoamerican efforts to other efforts worldwide.
ObjectivesMesoamerican nephropathy is an epidemic of chronic kidney disease (CKD) unrelated to traditional causes, mostly observed in sugarcane workers. We analysed CKD mortality in Costa Rica to ...explore when and where the epidemic emerged, sex and age patterns, and relationship with altitude, climate and sugarcane production.MethodsSMRs for CKD deaths (1970–2012) among population aged ≥20 were computed for 7 provinces and 81 counties over 4 time periods. Time trends were assessed with age-standardised mortality rates. We qualitatively examined relations between mortality and data on altitude, climate and sugarcane production.ResultsDuring 1970–2012, age-adjusted mortality rates in the Guanacaste province increased among men from 4.4 to 38.5 per 100 000 vs 3.6–8.4 in the rest of Costa Rica, and among women from 2.3 to 10.7 per 100 000 vs 2.6–5.0 in the rest of Costa Rica. A significant moderate excess mortality was observed among men in Guanacaste already in the mid-1970s, steeply increasing thereafter; a similar female excess mortality appeared a decade later, remaining stable. Male age-specific rates were high in Guanacaste for age categories ≥30, and since the late 1990s also for age range 20–29. The male spatiotemporal patterns roughly followed sugarcane expansion in hot, dry lowlands with manual harvesting.ConclusionsExcess CKD mortality occurs primarily in Guanacaste lowlands and was already present 4 decades ago. The increasing rates among Guanacaste men in hot, dry lowland counties with sugarcane are consistent with an occupational component. Stable moderate increases among women, and among men in counties without sugarcane, suggest coexisting environmental risk factors.
The classical nonhomologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The ...DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large DNA-PK catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (
) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (
) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in
and
mice because of the requirement for both hairpin opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056 cluster is the best-characterized autophosphorylation site in human DNA-PKcs. In this study, we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053. We also generated knockin mouse models with alanine- (DNA-PKcs
) or phospho-mimetic aspartate (DNA-PKcs
) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the
fibroblasts and lymphocytes, both
and
mice retained normal kinase activity and underwent efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053 cluster of murine DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.
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