The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

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.

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway.

The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.

Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks.

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair.

DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.

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.

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions.

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/µm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 µM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 µM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer) irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 µM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A) focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells.

The transcription factor TFII-I promotes DNA translesion synthesis and genomic stability.

Translesion synthesis (TLS) enables DNA replication through damaged bases, increases cellular DNA damage tolerance, and maintains genomic stability. The sliding clamp PCNA and the adaptor polymerase Rev1 coordinate polymerase switching during TLS. The polymerases Pol η, ι, and κ insert nucleotides opposite damaged bases. Pol ζ, consisting of the catalytic subunit Rev3 and the regulatory subunit Rev7, then extends DNA synthesis past the lesion. Here, we show that Rev7 binds to the transcription factor TFII-I in human cells. TFII-I is required for TLS and DNA damage tolerance. The TLS function of TFII-I appears to be independent of its role in transcription, but requires homodimerization and binding to PCNA. We propose that TFII-I bridges PCNA and Pol ζ to promote TLS. Our findings extend the general principle of component sharing among divergent nuclear processes and implicate TLS deficiency as a possible contributing factor in Williams-Beuren syndrome.

Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs.

Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment.

A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA-PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA-PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments.

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 interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair.

CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.

Dependence of Two-Photon eGFP Bleaching on Femtosecond Pulse Spectral Amplitude and Phase.

Photobleaching is a key limitation in two-photon imaging of fluorescent proteins with femtosecond pulsed excitation. We present measurements of the dependence of eGFP photobleaching on the spectral amplitude and phase of the pulses used. A strong dependence on the excitation wavelength was confirmed and measured over a 800-950 nm range. A fiber continuum light source and pulse shaping techniques were used to investigate photobleaching with broadband, 15 fs transform limited, pulses with differing spectral amplitude and phase. Narrow band pulses, >150 fs transform limited, typical of femtosecond laser sources used in two-photon imaging applications, were also investigated for their photobleaching dependence on pulse dispersion and bandwidth. The bleach rate for broadband pulses was found to be primarily determined by the second harmonic spectrum of the excitation light. On the other hand, for narrow band excitation pulses with similar center wavelengths improvement in bleach rate was found to be mostly dependent on reducing the pulse length. A simple model to predict the relative bleach rates for broadband pulses is presented and compared to the experimental data.

Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells.

The concept of DNA "repair centers" and the meaning of radiation-induced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 µm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF/Gy after 2 Gy exposure compared to approximately 64 RIF/Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation.

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.

Unrepaired clustered DNA lesions induce chromosome breakage in human cells.

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis.

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.