NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor-in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor-functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head-associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1's interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.

Dysfunctional telomeres trigger cellular senescence mediated by cyclic GMP-AMP synthase.

Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double strand breaks specifically in the telomeres, ChIP, telomere immunofluorescence, fluorescence in situ hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.

RAD51 interconnects between DNA replication, DNA repair and immunity.

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.

Ionizing radiation and heart risks.

Cardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. As advancements in radiation therapy (RT) have significantly increased the number of cancer survivors, the risk of radiation-induced cardiovascular disease (RICD) in this group is a growing concern. Recent epidemiological data suggest that accidental or occupational exposure to low dose radiation, in addition to therapeutic ionizing radiation, can result in cardiovascular complications. The progression of radiation-induced cardiotoxicity often takes years to manifest but is also multifaceted, as the heart may be affected by a variety of pathologies. The risk of cardiovascular disease development in RT cancer survivors has been known for 40 years and several risk factors have been identified in the last two decades. However, most of the early work focused on clinical symptoms and manifestations, rather than understanding cellular processes regulating homeostatic processes of the cardiovascular system in response to radiation. Recent studies have suggested that a different approach may be needed to refute the risk of cardiovascular disease following radiation exposure. In this review, we will focus on how different radiation types and doses may induce cardiovascular complications, highlighting clinical manifestations and the mechanisms involved in the pathophysiology of radiation-induced cardiotoxicity. We will finally discuss how current and future research on heart development and homeostasis can help reduce the incidence of RICD.

Werner Syndrome Protein and DNA Replication.

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3' to 5' helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN's contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN's molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

Mechanistic link between DNA damage sensing, repairing and signaling factors and immune signaling.

Previously, DNA damage sensing, repairing and signaling machineries were thought to mainly suppress genomic instability in response to genotoxic stress. Emerging evidence indicates a crosstalk between DNA repair machinery and the immune system. In this chapter, we attempt to decipher the molecular choreography of how factors, including ATM, BRCA1, DNA-PK, FANCA/D2, MRE11, MUS81, NBS1, RAD51 and TREX1, of multiple DNA metabolic processes are directly or indirectly involved in suppressing cytosolic DNA sensing pathway-mediated immune signaling. We provide systematic details showing how different DDR factors' roles in modulating immune signaling are not direct, but are rather a consequence of their inherent ability to sense, repair and signal in response to DNA damage. Unexpectedly, most DDR factors negatively impact the immune system; that is, the immune system shows defective signaling if there are defects in DNA repair pathways. Thus, in addition to their known DNA repair and replication functions, DDR factors help prevent erroneous activation of immune signaling. A more precise understanding of the mechanisms by which different DDR factors function in immune signaling can be exploited to redirect the immune system for both preventing and treating autoimmunity, cellular senescence and cancer in humans.

Repurposing DNA repair factors to eradicate tumor cells upon radiotherapy.

Cancer is the leading cause of death worldwide. Almost 50% of all cancer patients undergo radiation therapy (RT) during treatment, with varying success. The main goal of RT is to kill tumor cells by damaging their DNA irreversibly while sparing the surrounding normal tissue. The outcome of RT is often determined by how tumors recognize and repair their damaged DNA. A growing body of evidence suggests that tumors often show abnormal expression of DNA double-strand break (DSB) repair genes that are absent from normal cells. Defects in a specific DNA repair pathway make tumor cells overly dependent on alternative or backup pathways to repair their damaged DNA. These tumor cell-specific abnormalities in the DNA damage response (DDR) machinery can potentially be used as biomarkers for treatment outcomes or as targets for sensitization to ionizing radiation (IR). An improved understanding of genetic or epigenetic alterations in the DNA repair pathways specific to cancer cells has paved the way for new treatments that combine pharmacological exploitation of tumor-specific molecular vulnerabilities with IR. Inhibiting DNA repair pathways has the potential to greatly enhance the therapeutic ratio of RT. In this review, we will discuss DNA repair pathways in active cells and how these pathways are deregulated in tumors. We will also describe the impact of targeting cancer-specific aberrations in the DDR as a treatment strategy to improve the efficacy of RT. Finally, we will address the current roadblocks and future prospects of these approaches.

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway.

Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability.

Human ventricular unloading induces cardiomyocyte proliferation.

BACKGROUND: The adult mammalian heart is incapable of meaningful regeneration after substantial cardiomyocyte loss, primarily due to the inability of adult cardiomyocytes to divide. Our group recently showed that mitochondria-mediated oxidative DNA damage is an important regulator of postnatal cardiomyocyte cell cycle arrest. However, it is not known whether mechanical load also plays a role in this process. We reasoned that the postnatal physiological increase in mechanical load contributes to the increase in mitochondrial content, with subsequent activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes. OBJECTIVES: The purpose of this study was to test the effect of mechanical unloading on mitochondrial mass, DDR, and cardiomyocyte proliferation. METHODS: We examined the effect of human ventricular unloading after implantation of left ventricular assist devices (LVADs) on mitochondrial content, DDR, and cardiomyocyte proliferation in 10 matched left ventricular samples collected at the time of LVAD implantation (pre-LVAD) and at the time of explantation (post-LVAD). RESULTS: We found that post-LVAD hearts showed up to a 60% decrease in mitochondrial content and up to a 45% decrease in cardiomyocyte size compared with pre-LVAD hearts. Moreover, we quantified cardiomyocyte nuclear foci of phosphorylated ataxia telangiectasia mutated protein, an upstream regulator of the DDR pathway, and we found a significant decrease in the number of nuclear phosphorylated ataxia telangiectasia mutated foci in the post-LVAD hearts. Finally, we examined cardiomyocyte mitosis and cytokinesis and found a statistically significant increase in both phosphorylated histone H3-positive, and Aurora B-positive cardiomyocytes in the post-LVAD hearts. Importantly, these results were driven by statistical significance in hearts exposed to longer durations of mechanical unloading. CONCLUSIONS: Prolonged mechanical unloading induces adult human cardiomyocyte proliferation, possibly through prevention of mitochondria-mediated activation of DDR.

Nonenzymatic role for WRN in preserving nascent DNA strands after replication stress.

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.