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Poly (ADP-ribose) polymerase-1 (PARP1) and 2 (PARP2) are two DNA damage-induced poly (ADP-ribose) (PAR) polymerases in cells and are the targets of PARP inhibitors used for cancer therapy. Strand breaks recruit and activate PARP1 and 2, which rapidly generate PAR from NAD+. PAR promotes the recruitment of other repair factors, relaxes chromatin, and has a role in DNA repair, transcription regulation, and RNA biology. Four PARP1/2 dual inhibitors are currently used to treat BRCA-deficient breast, ovarian, prostate, and pancreatic cancers. In addition to blocking the enzymatic activity of PARP1 and 2, clinical PARP inhibitors extend the appearance of PARP1 and PARP2 on chromatin after damage, termed trapping. Loss of PARP1 confers resistance to PARP inhibitors, suggesting an essential role of trapping in cancer therapy. Yet, whether the persistent PARP1 and 2 foci at the DNA damage sites are caused by the retention of the same molecules or by the continual exchange of different molecules remains unknown. Here, we discuss recent results from quantitative live-cell imaging studies focusing on PARP1 and PARP2's distinct DNA substrate specificities and modes of recruitment and trapping with implications for cancer therapy and on-target toxicities of PARP inhibitors.

PARP1 is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A,Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1Parp1+/Amice are chimeras with mixedParp1+/AN(neoR retention) cells that act similarly toParp1+/-. Pure F2Parp1+/Aembryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared toParp1-/-cells, genotype and expression-validated pureParp1+/Acells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatin exchange and mitotic bridges. Despite proficiency for homologous recombination and non-homologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development,Parp1+/Acells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity ofParp1+/Acells to base damages and Topo inhibitors in particular exceedParp1-/-controls. The findings show that the enzymatically inactive PARP1 protein has a dominant negative role and establishes a clear physiological difference between PARP1 inactivation vs. deletion. As a result, the enzymatically inactive PARP1 has a much more deteriorating impact on normal tissues than previously estimated, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.Significance StatementPARP1 is the primary target of PARP enzymatic inhibitors. The use of PARP inhibitors for cancer therapy is based not only on the extreme sensitivity of BRCA1/2-deficient cancer cells to PARP1 inhibition but also on the nonessential role of PARP1 in normal tissues. Here we show that in contrast to the normal development of Parp1-null mice, the mouse model expressing the catalytically inactive Parp1 on only one allele (E988A,Parp1+/A) dies embryonically with high levels of genomic instability. The results reveal the severe dominant-negative impact of catalytically inactive PARP1, indicating the presence of enzymatically inactive PARP1 is much more damaging to normal tissues than previously anticipated. These findings provide a mechanism for clinical PARP inhibitors’ unexpected normal tissue toxicity.

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm−/− controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations.Significant:This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.

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Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptor (TLR) and IL-1R signaling, with no reported roles outside of innate immunity. We find that vertebrate cells exposed to ionizing radiation (IR) sequentially activate IRAK4 and IRAK1 through a phosphorylation cascade mirroring that induced by TLR/IL-1R, resulting in a potent anti-apoptotic response. However, IR-induced IRAK1 activation does not require the receptors or the IRAK4/1 adaptor protein MyD88, and instead of remaining in the cytoplasm, the activated kinase is immediately transported to the nucleus via a conserved nuclear localization signal. We identify: double-strand DNA breaks (DSBs) as the biologic trigger for this pathway; the E3 ubiquitin ligase Pellino1 as the scaffold enabling IRAK4/1 activation in place of TLR/IL-1R-MyD88; and the pro-apoptotic PIDDosome (PIDD1-RAIDD-caspase-2) as a critical downstream target in the nucleus. The data delineate a non-canonical IRAK signaling pathway derived from, or ancestral to, TLR signaling. This DSB detection pathway, which is also activated by genotoxic chemotherapies, provides multiple actionable targets for overcoming tumor resistance to mainstay cancer treatments.

PARP1 and PARP2 detect DNA breaks, which activates their catalytic production of poly(ADP-ribose) that recruits repair factors and releases PARP1/2 from DNA. PARP inhibitors (PARPi) are used in cancer treatment and target PARP1/2 catalytic activity, interfering with repair and increasing PARP1/2 persistence on DNA damage. Additionally, certain PARPi exert allosteric effects that increase PARP1 retention on DNA. However, no clinical PARPi exhibit this allosteric behavior toward PARP1. In contrast, we show that certain clinical PARPi exhibit an allosteric effect that retains PARP2 on DNA breaks in a manner that depends on communication between the catalytic and DNA-binding regions. Using a PARP2 mutant that mimics an allosteric inhibitor effect, we observed increased PARP2 retention at cellular damage sites. The new PARPi AZD5305 also exhibited a clear reverse allosteric effect on PARP2. Our results can help explain the toxicity of clinical PARPi and suggest ways to improve PARPi moving forward.

Recent structural studies have revealed the atomic details of how the DNA-PKcs kinase protects DNA ends, recruits and activates Artemis endonuclease for end-processing, and phosphorylates itself for end-ligation, revealing the molecular details from end-processing to end-ligation, two critical phases of the non-homologous end-joining (NHEJ) DNA-double strand break repair pathway.

Nuclear actin-based movements have been shown to orchestrate clustering of DNA double-strand breaks (DSBs) into homology-directed repair domains. Here we describe multiscale three-dimensional genome reorganization following DNA damage and analyze the contribution of the nuclear WASP-ARP2/3-actin pathway toward chromatin topology alterations and pathologic repair. Hi-C analysis reveals genome-wide, DNA damage-induced chromatin compartment flips facilitated by ARP2/3 that enrich for open, A compartments. Damage promotes interactions between DSBs, which in turn facilitate aberrant, actin-dependent intra- and inter-chromosomal rearrangements. Our work establishes that clustering of resected DSBs into repair domains by nuclear actin assembly is coordinated with multiscale alterations in genome architecture that enable homology-directed repair while also increasing nonhomologous end-joining-dependent translocation frequency.

Summary Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1−/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity., Graphical abstract, Highlights • XRCC1 prevents endogenous PARP1 trapping during DNA base excision repair • PARP1 trapping impedes base excision repair and increases sensitivity to base damage • In the absence of PARP1, XRCC1 is dispensable for DNA base excision repair, Demin et al. show that the essential role of the scaffold protein XRCC1 during DNA base excision repair is to prevent toxic “trapping” of PARP1 on SSB intermediates, which otherwise block this essential repair process and lead to increased cellular sensitivity to DNA base damage.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm−/− controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations. Significant: This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

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) factor1. 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 cNHEJ2. 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 lymphoma3. 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 rRNA4. 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.

Dual-inhibitors of PARP1 and PARP2 are promising anti-cancer drugs. In addition to blocking PARP1&2 enzymatic activity, PARP inhibitors also extend the lifetime of DNA damage-induced PARP1&2 foci, termed trapping. Trapping is important for the therapeutic effects of PARP inhibitors. Using live-cell imaging, we found that PARP inhibitors cause persistent PARP2 foci by switching the mode of PARP2 recruitment from a predominantly PARP1- and PAR-dependent rapid exchange to a WGR domain-mediated stalling of PARP2 on DNA. Specifically, PARP1-deletion markedly reduces but does not abolish PARP2 foci. The residual PARP2 foci in PARP1-deficient cells are DNA-dependent and abrogated by the R140A mutation in the WGR domain. Yet, PARP2-R140A forms normal foci in PARP1-proficient cells. In PARP1-deficient cells, PARP inhibitors - niraparib, talazoparib, and, to a lesser extent, olaparib - enhance PARP2 foci by preventing PARP2 exchange. This trapping of PARP2 is independent of auto-PARylation and is abolished by the R140A mutation in the WGR domain and the H415A mutation in the catalytic domain. Taken together, we found that PARP inhibitors trap PARP2 by physically stalling PARP2 on DNA via the WGR-DNA interaction while suppressing the PARP1- and PAR-dependent rapid exchange of PARP2.

A scalable co-assay for single cell mRNA and gDNA sequencing could potentially harmonize the identification of rare cell populations and cell states with corresponding cell-lineage relationships, drug sensitivities, and genetic mutations. Such an assay would provide new insights into the relationship between genetic alterations and tumor cell phenotype. However, simultaneously measuring the genomic and transcriptomic content of individual cells at high throughput remains challenging. Here we present single-cell lithium assisted nucleosome depleted sequencing (scLAND-seq), a scalable method for co-sequencing RNA and DNA from single nuclei that uses the widely available 10x Genomics microfluidics platform. In scLAND-seq, nuclei are treated with a mild detergent, lithium diiodosalicylate, which denatures protein and renders gDNA free of nucleosome packing while preserving the nuclear membrane. The nuclei are then tagmented with Tn5 transposase, which randomly fragments gDNA and integrates universal sequencing adapters. Tagmented nuclei are loaded into a microfluidic droplet generator which co-encapsulates nuclei with beads containing barcoded primers for reverse transcription of mRNA and replication of gDNA. Ultimately two libraries are created: one for nuclear mRNA and one for gDNA. Each library contains barcodes linking it to its nuclei of origin, thus allowing simultaneous analysis of the transcriptome and genome of each nucleus. Once nuclei have been depleted of nucleosomes, all steps can be performed using a 10x Genomics Chromium Controller and 10x Multiome Kit without further experimental modification. We characterize LAND-seq with BJ fibroblasts, a human diploid cell line, and demonstrate the detection of rare subpopulations with copy number variants (CNVs) associated with specific transcriptional clusters. Analysis of transcription start site enrichment indicates near-complete disruption of chromatin structure, resulting in high-complexity libraries that could not be sequenced to saturation even with >1M reads/cell. The corresponding gene expression library is of high-quality, containing ~20,000 gene counts per nuclei. Finally, to demonstrate scLAND-seq’s compatibility with primary cells, we applied scLAND-seq to cryopreserved, human glioblastoma (GBM) surgical specimens, enabling direct investigation into the relationship between phenotype and genotype in thousands of individual cells. Character limit: 2,600 Citation Format: Timothy R. Olsen, Pranay Talla, Julia Furnari, Shan Zha, Peter Canoll, Jeffrey Bruce, Peter Sims. Scalable co-sequencing of RNA and DNA from individual nuclei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1170.

SummaryDNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

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 (DNA-PKcs−/−) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs−/− and DNA-PKcsKD/KD 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-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD 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.

The DNA damage response protein ATM has long been known to influence class switch recombination in ex vivo–cultured B cells. However, an assessment of B cell–intrinsic requirement of ATM in humoral responses in vivo was confounded by the fact that its germline deletion affects T cell function, and B:T cell interactions are critical for in vivo immune responses. In this study, we demonstrate that B cell–specific deletion of ATM in mice leads to reduction in germinal center (GC) frequency and size in response to immunization. We find that loss of ATM induces apoptosis of GC B cells, likely due to unresolved DNA lesions in cells attempting to undergo class-switch recombination. Accordingly, suboptimal GC responses in ATM-deficient animals are characterized by decreased titers of class-switched Abs and decreased rates of somatic hypermutation. These results unmask the critical B cell–intrinsic role of ATM in maintaining an optimal GC response following immunization.

Genomic integrity is critical for normal development, healthy aging and suppressing oncogenic transformation. The DNA damage response (DDR) is a complex network that is activated by DNA structural changes to preserve genome integrity. Situated at the apex of the mammalian DDR are three PI3-kinase-related protein kinases—ATM, DNA-PKcs and ATR. They are activated by different DNA lesions via direct binding to their unique sensor protein complexes (MRE11-RAD50-NBS1 for ATM, Ku70-Ku80/86 for DNA-PKcs and ATRIP-RPA for ATR) and phosphorylate a large number of partially overlapping substrates, including themselves and each other to promote DNA repair and regulate cell cycle checkpoints and tissue homeostasis. This review focuses on mouse models with deletion and point mutations of ATM, DNA-PKcs and ATR, and discusses how their activation mechanism and their kinase activity contribute to their unique, yet interactive roles in DNA repair in general and during tissue-specific development processes and how their deficiency leads to specific physiological and pathophysiological consequences.

Evapotranspiration (ET) is an important part of water cycle and energy flow in ecosystem. Accurate estimation of ET and its components is critical for understanding the impacts of ecophysiological processes on ecosystem water balance and plant water use strategy. Using the eddy-covariance technique and the micro-lysimeter, we measured ET, evaporation (蒸散发(ET)是生态系统水分循环和能量流动的重要组成部分,准确估算ET及其各组分,对认识生态生理过程对水分平衡和植物水分利用策略的影响具有重要意义。本研究于2019年5月20日至9月15日,利用涡度相关技术和微型蒸渗仪对毛乌素沙地油蒿-杨柴灌丛生态系统ET、蒸发(

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4(−/−)Tp53(−/−) mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs(−/−)Tp53(−/−) mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKIN) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here we report the cryoEM structures of DNA-PKcs (catalytic subunit) bound to a DNA end, or complexed with Ku70/80 and DNA, in both inactive and activated forms at resolutions of 3.7 Å overall, and 3.2 Å for FATKIN. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.

Forests play an important role in terrestrial carbon cycles. The mechanism underlying carbon balance in temperate deciduous broad-leaved forests is not clear. In this study, net ecosystem exchange (NEE) and environmental factors, including air temperature (森林生态系统在陆地碳循环过程中发挥着重要作用,关于温带落叶阔叶林生态系统碳平衡过程影响机制的讨论尚未统一。本研究于2019年对北京松山典型落叶阔叶林生态系统的净碳交换量(NEE)及空气温度(

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as ‘trapping’. To understand the molecular nature of ‘trapping’ in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

The DNA-dependent protein kinase (DNA-PK), composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous 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. DNA-PKcs is the best-characterized substrate of DNA-PK, which phosphorylates DNA-PKcs at both the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity, suggesting a role of T2609 phosphorylation in DNA repair. Using the DNA-PKcs5A mouse model carrying an alanine substitution 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/5A B cells reveal increased chromosomal translocation, excess end-resection, and preferential usage of micro-homology – 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.Key pointsLoss of T2069 cluster phosphorylation of DNA-PKcs promotes Alt-EJ-mediated CSR.

Ataxia-telangiectasia mutated (ATM) kinase is a master regulator of the DNA damage response, and loss of ATM leads to primary immunodeficiency and greatly increased risk for lymphoid malignancies. The FATC domain is conserved in phosphatidylinositol-3-kinase–related protein kinases (PIKKs). Truncation mutation in the FATC domain (R3047X) selectively compromised reactive oxygen species–induced ATM activation in cell-free assays. In this article, we show that in mouse models, knock-in ATM-R3057X mutation (Atm⁠RX⁠, corresponding to R3047X in human ATM) severely compromises ATM protein stability and causes T cell developmental defects, B cell Ig class-switch recombination defects, and infertility resembling ATM-null. The residual ATM-R3057X protein retains minimal yet functional measurable DNA damage-induced checkpoint activation and significantly delays lymphomagenesis in Atm⁠RX/RX⁠ mice compared with Atm⁠−/−⁠. Together, these results support a physiological role of the FATC domain in ATM protein stability and show that the presence of minimal residual ATM-R3057X protein can prevent growth retardation and delay tumorigenesis without restoring lymphocyte development and fertility.

Radiation dosimeters are critical for accurately assessing the levels of radiation exposure of tumor sites and surrounding tissues and for optimizing therapeutic interventions as well as for monitoring environmental exposure. To fill the need for a simple, user-friendly, and inexpensive dosimeter, we designed an innovative colorimetric nanosensor-based assay for detecting ionizing radiation. We show that hydroxyl radicals generated by ionizing radiation can be used to etch gold nanorods (AuNRs) and silver nanoprisms (AgNPRs), yielding reproducible color changes for radiation dose detection in the range of 50–2000 rad, broad enough to cover doses used in hyperfractionated, conventional, and hypofractionated radiotherapy. This range of doses detected by this assay correlates with radiation-induced DNA damage response in mammalian cells. Furthermore, this AuNR- and AgNPR-based sensing platform has been established in a paper format that can be readily adopted for a wide range of applications and translation.

Revealing the seasonal and interannual variations in leaf-level photosynthesis is a critical issue in understanding the ecological mechanisms underlying the dynamics of carbon dioxide exchange between the atmosphere and shrub ecosystem. Artemisia ordosica is a dominant shrub species in semi-arid area of northwest China. Photosynthetic gas exchange, leaf nitrogen content(LN), specific leaf area (SLA) and some environmental factors were measured simultaneously on clear days (rotated every 10 days) of the growing season from 2011 to 2018, to quantify the temporal variations and environmental controls of photosynthetic parameters. Our results demonstrated that mean value of light-response curve parameters, the maximum photosynthetic capacity (Pmax), appear quality efficiency (AQE), respiration in dark (Rd), light saturated point (LSP) and light compensated point (LCP) had a gradual decline with the growth (spring> summer>autumn). Structural equation modeling (SEM) was used to elucidate the direct and indirect effects of biophysical factors on Pmax. The driven factors of Pmax in growing season changed, but stomatal conductance (gs) was the dominant factor in all stages. The gs was influenced by SLA and LN，and the soil water content at a depth of 10cm (SWC10) affected the Pmax in spring. In summer, Pmax was significantly positively related with gs and transpiration rate (Tr), and gs was influenced by SLA, LN and soil water content at a depth of 30cm (SWC30). In autumn, Pmax was significantly positively correlated with gs, while was significantly negatively correlated with air temperature (Ta). This simulation based on situ ecophysiological research suggest that Pmax of A. ordosica responded to the environment factors of seasonal and interannual variations, which is not the inherent genetic characteristics. Soil water content is the major environmental factor influencing Pmax in spring and summer, while Ta is the major one in autumn. Knowledge of how environmental change will affect the photosynthesis of A. ordosica in the future is essential for their protection, adaptation strategies and carbon fixation prediction in shrub ecosystems.

DNA damage, especially DNA double strand breaks (DSBs) and replication stress, activates a complex post-translational network termed DNA damage response (DDR). Our review focuses on three PI3-kinase related protein kinases—ATM, ATR and DNA-PKcs, which situate at the apex of the mammalian DDR. They are recruited to and activated at the DNA damage sites by their respective sensor protein complexes—MRE11/RAD50/NBS1 for ATM, RPA/ATRIP for ATR and KU70–KU80/86 (XRCC6/XRCC5) for DNA-PKcs. Upon activation, ATM, ATR and DNA-PKcs phosphorylate a large number of partially overlapping substrates to promote efficient and accurate DNA repair and to coordinate DNA repair with other DNA metabolic events (e.g., transcription, replication and mitosis). At the organism level, robust DDR is critical for normal development, aging, stem cell maintenance and regeneration, and physiological genomic rearrangements in lymphocytes and germ cells. In addition to endogenous damage, oncogene-induced replication stresses and genotoxic chemotherapies also activate DDR. On one hand, DDR factors suppress genomic instability to prevent malignant transformation. On the other hand, targeting DDR enhances the therapeutic effects of anti-cancer chemotherapy, which led to the development of specific kinase inhibitors for ATM, ATR and DNA-PKcs. Using mouse models expressing kinase dead ATM, ATR and DNA-PKcs, an unexpected structural function of these kinases was revealed, where the expression of catalytically inactive kinases causes more genomic instability than the loss of the proteins themselves. The spectrum of genomic instabilities and physiological consequences are unique for each kinase and depends on their activating complexes, suggesting a model in which the catalysis is coupled with DNA/chromatin release and catalytic inhibition leads to the persistence of the kinases at the DNA lesion, which in turn affects repair pathway choice and outcomes. Here we discuss the experimental evidences supporting this mode of action and their implications in the design and use of specific kinase inhibitors for ATM, ATR and DNA-PKcs for cancer therapy.

Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCL) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2 positive DLBCL and T cell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to Poly (ADP-ribose) polymerase (PARP) inhibitors. Further, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2 positive tumors. Together, our results reveal that LMO2 expression predicts HR-deficiency and the potential therapeutic utility of PARP inhibitors in DLBCL and T-ALL.

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

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice.

Lymphocyte development requires ordered assembly and subsequent modifications of the antigen receptor genes through V(D)J recombination and Immunoglobulin class switch recombination (CSR), respectively. While the programmed DNA cleavage events are initiated by lymphocyte-specific factors, the resulting DNA double-strand break (DSB) intermediates activate the ATM kinase-mediated DNA damage response (DDR) and rely on the ubiquitously expressed classical non-homologous end-joining (cNHEJ) pathway including the DNA-dependent protein kinase (DNA-PK), and, in the case of CSR, also the alternative end-joining (Alt-EJ) pathway, for repair. Correspondingly, patients and animal models with cNHEJ or DDR defects develop distinct types of immunodeficiency reflecting their specific DNA repair deficiency. The unique end-structure, sequence context, and cell cycle regulation of V(D)J recombination and CSR also provide a valuable platform to study the mechanisms of, and the interplay between, cNHEJ and DDR. Here, we compare and contrast the genetic consequences of DNA repair defects in V(D)J recombination and CSR with a focus on the newly discovered cNHEJ factors and the kinase-dependent structural roles of ATM and DNA-PK in animal models. Throughout, we try to highlight the pending questions and emerging differences that will extend our understanding of cNHEJ and DDR in the context of primary immunodeficiency and lymphoid malignancies.

ATR kinase is activated by RPA-coated single-stranded DNA (ssDNA) to orchestrate DNA damage responses. Here we show that ATR inhibition differs from ATR loss. Mouse model expressing kinase-dead ATR (Atr+/KD), but not loss of ATR (Atr+/−), displays ssDNA-dependent defects at the non-homologous region of X-Y chromosomes during male meiosis leading to sterility, and at telomeres, rDNA, and fragile sites during mitosis leading to lymphocytopenia. Mechanistically, we find that ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATR-KD, but not loss of ATR, traps a subset of ATR and RPA on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-PKcs and prevents downstream repair. Consequently, Atr+/KD cells have shorter inter-origin distances and are vulnerable to induced fork collapses, genome instability and mitotic catastrophe. These results reveal mechanistic differences between ATR inhibition and ATR loss, with implications for ATR signaling and cancer therapy., ATR kinase is a key regulator of chromosome integrity. Here the authors by analysing the phenotype of a mouse model expressing a kinase-dead ATR, reveal the effect of ATR inhibition compared to ATR loss and its consequences for meiosis, DNA replication, checkpoint activation and genome instability .

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcs(KD/KD)) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcs(KD/KD), but not DNA-PKcs(−/−), B cells. Meanwhile, the residual joints from DNA-PKcs(KD/KD) cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs(−/−) B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs(−/−) cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.

In arid and semi-arid areas, plant physiological responses to water availability depend largely on the intensity and frequency of rain events. Knowledge on the responses of xerophytic plants to rain events is important for predicting the structure and functioning of dryland ecosystems under changing climate. The sap flow of Hedysarum scoparium in the Mu Us Sand Land was continuously measured during the growing season of 2012 and 2013. The objectives were to quantify the dynamics of sap flow under different weather conditions, and to examine the responses of sap flow to rain events of different sizes. The results showed that the daily sap flow rates of H. scoparium were lower on rainy days than on clear days. On clear days, the sap flow of H. scoparium showed a midday plateau, and was positively correlated with solar radiation and relative humidity. On rainy days, the sap flow fluctuated at low levels, and was positively correlated with solar radiation and air temperature. Rain events not only affected the sap flow on rainy days through variations in climatic factors (e.g., solar radiation and air temperature), but also affected post-rainfall sap flow velocities though changes in soil moisture. Small rain events (20 mm) did not change the sap flow, whereas large rain events (20 mm) significantly increased the sap flow on days following rainfall. Rain-wetted soil conditions not only resulted in higher sap flow velocities, but also enhanced the sensitivity of sap flow to solar radiation, vapor pressure deficit and air temperature.干旱半干旱区植物生理过程的水分响应依赖于降雨强度和频率,研究典型沙生植物对降雨的响应有助于预测未来气候条件下荒漠生态系统的结构和功能变化.2012和2013年花棒生长季,在宁夏盐池采用包裹式茎流仪对花棒茎流进行连续观测,分析茎流在晴天和雨天的变化特征及其对不同降雨事件的响应.结果表明: 雨天的花棒日茎流量低于晴天.晴天,茎流日变化呈“几”字型宽峰曲线,与太阳辐射和相对湿度呈正相关;雨天,茎流日变化呈多峰曲线或者一直处于极低状态,与太阳辐射和空气温度呈正相关.降雨不仅可以通过影响当天太阳辐射、空气温度、饱和蒸汽压差和相对湿度影响花棒茎流通量,还通过调控土壤含水量影响降雨后的茎流通量.降雨量20 mm时,降雨前后茎流通量差异不显著;降雨量20 mm时,降雨后的茎流通量比降雨前有显著提高.高土壤含水量不仅能够提高茎流通量,并且能够提高茎流通量对太阳辐射、饱和水汽压差和空气温度的响应敏感性.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell malignancy. Here we integrated large-scale profiling data of alterations in gene expression, allelic copy number (CN), and nucleotide sequences in 111 well-characterized patients. Besides prominent signatures of T-cell activation and prevalent clonal variants, we also identify novel hot-spots for CN variability, fusion molecules, alternative transcripts, and progression-associated dynamics. The overall lesional spectrum of T-PLL is mainly annotated to axes of DNA damage responses, T-cell receptor/cytokine signaling, and histone modulation. We formulate a multi-dimensional model of T-PLL pathogenesis centered around a unique combination of TCL1 overexpression with damaging ATM aberrations as initiating core lesions. The effects imposed by TCL1 cooperate with compromised ATM toward a leukemogenic phenotype of impaired DNA damage processing. Dysfunctional ATM appears inefficient in alleviating elevated redox burdens and telomere attrition and in evoking a p53-dependent apoptotic response to genotoxic insults. As non-genotoxic strategies, synergistic combinations of p53 reactivators and deacetylase inhibitors reinstate such cell death execution., T-cell prolymphocytic leukemia (T-PLL) is a rare malignancy with a poor prognosis. Here, the authors investigate the genomic landscape, gene expression profiles and functional mechanisms in 111 patients, highlighting TCL1 overexpression and ATM aberrations as core lesions which co-operate to impair DNA damage processing.

The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1(S563F) and Bard1(K607A)) or Brca1 (Brca1(S1598F)). Brca1(S1598F) abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1-mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1(S563F/S563F) and Bard1(K607A/K607A) mice, unlike Brca1(S1598F/S1598F) mice, are not tumor-prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, since SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1-mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.

During the last decade, the widely distributed shrublands in northern China have shown significant signs of recovery from desertification, the result of widespread conservation practices. However, to support the current efforts in conservation, more knowledge is needed on surface energy partitioning and its biophysical controls. Using eddy-covariance measurements made over a semi-arid shrubland in northwest China in 2012, we examined how surface energy-balance components vary on diurnal and seasonal scales, and how biophysical factors control bulk surface parameters and energy exchange. Sensible heat flux (H) exceeded latent heat flux (λE) during most of the year, resulting in an annual Bowen ratio (β, i.e. H/λE) of 2.0. λE exceeded H only in mid-summer when frequent rainfall co-occurred with the seasonal peak in leaf area index (LAI). Evapotranspiration reached a daily maximum of 3.3 mm day−1, and summed to 283 mm yr−1. The evaporative fraction (EF, i.e. λE/Rn), Priestley–Taylor coefficient (α), surface conductance (gs) and decoupling coefficient (Ω) were all positively correlated with soil water content (SWC) and LAI. The direct enhancement of λE by high vapour pressure deficit (VPD) was buffered by a concurrent suppression of gs. The gs played a direct role in controlling EF and α by mediating the effects of LAI, SWC and VPD. Our results highlight the importance of adaptive plant responses to water scarcity in regulating ecosystem energy partitioning, and suggest an important role for revegetation in the reversal of desertification in semi-arid areas. Copyright © 2015 John Wiley & Sons, Ltd.

Ataxia telangiectasia mutated (ATM) is a protein kinase and a master regulator of DNA-damage responses. Germline ATM inactivation causes ataxia-telangiectasia (A-T) syndrome with severe lymphocytopenia and greatly increased risk for T-cell lymphomas/leukemia. Both A-T and T-cell prolymphoblastic leukemia patients with somatic mutations of ATM frequently carry inv(14;14) between the T-cell receptor α/δ (TCRα/δ) and immunoglobulin H loci, but the molecular origin of this translocation remains elusive. ATM(-/-) mice recapitulate lymphocytopenia of A-T patients and routinely succumb to thymic lymphomas with t(12;14) translocation, syntenic to inv(14;14) in humans. Here we report that deletion of the TCRδ enhancer (Eδ), which initiates TCRδ rearrangement, significantly improves αβ T cell output and effectively prevents t(12;14) translocations in ATM(-/-) mice. These findings identify the genomic instability associated with V(D)J recombination at the TCRδ locus as the molecular origin of both lymphocytopenia and the signature t(12;14) translocations associated with ATM deficiency.

Non-homologous 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 auto-phosphorylated or trans-phosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated trans-phosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strictly auto-phosphorylation 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.