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

Abstract

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.

1. Introduction

DNA damage is a biological process that negatively impacts human health in many ways. Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities, such as DNA replication and recombination errors, or environmental exposures, such as ionizing radiation, ultraviolet light and chemical mutagens. To ensure genomic integrity, cells have evolved sophisticated mechanisms to repair DNA damage, including DNA double-strand breaks (DSBs). The two major DSB repair pathways are non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ is an error-prone repair pathway that can occur throughout all cell cycle phases, whereas HR is an error-free pathway that predominantly occurs in late S and G₂ phases. Alterations in the pathways involved in repairing DSB and processing stalled or collapsed replication forks cause either cellular senescence, immune disorder or cancer.

In the case of HR repair, DSBs are first recognized by the MRN complex, made up of MRE11, RAD50 and NBS1, which induces the auto-phosphorylation of the ATM (ataxia telangiectasia-mutated) kinase (Lamarche, Orazio, & Weitzman, 2010). Phosphorylation of ATM then leads to the activation, phosphorylation and amplification of a cascade of downstream effector proteins, including phosphorylation of H2AX, the kinase CHK2 and the cohesin protein SMC1 (Kitagawa, Bakkenist, McKinnon, & Kastan, 2004). These factors colocalize to distinct nuclear repair foci. Additional repair proteins, such as 53BP1, BRCA1/2, NBS1 and RAD51, are also recruited to these sites, which leads to DNA resection and faithful homologous recombination repair in the S/G₂ phase (Fradet-Turcotte, Sitz, Grapton, & Orthwein, 2016; Niu et al., 2010). The ATR (ataxia telangiectasia and Rad3-related protein) DNA-related pathway is activated in response to single-strand DNA breaks but can augment ATM activity and is mediated through the CHK1 kinase (Cimprich & Cortez, 2008). In contrast, NHEJ uses different sets of factors and is prone to error (Chapman, Taylor, & Boulton, 2012).

In response to DNA breaks, cells initiate a series of signaling pathways, namely DNA damage response (DDR) signaling, mediated by members of the phosphatidylinositol 3-kinase (PI3K)-like kinase (PIKK) family, ATM, ATR and DNA-PK (DNA-dependent kinase). ATM plays a critical role in DNA damage signaling originating at DSBs, whereas ATR responds to single-strand DNA (ssDNA) regions associated with replication forks. DNA-PK is directly involved in DNA repair by promoting DSB religation through NHEJ. DDR signaling contributes to DNA break repair, transient cell cycle arrest, and transcriptional and post-transcriptional activation of a wide array of genes, and, under certain circumstances, it triggers programmed cell death. Faithful repair of DNA lesions and activation of DDR signaling are critical for cellular survival and preventing genomic instability and premature cellular senescence.

The pattern recognition receptors (PRR) of the innate immune system are the first line of host defense, as they recognize the various pathogen- or danger-associated molecular patterns and elicit defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-8 or interferon β (IFN-β). Of the many factors that participate in immune signaling in response to DNA in the cytosol, cyclic GMP-AMP synthase (cGAS) is one of the most essential. cGAS is a cytosolic DNA sensor/enzyme that catalyzes the formation of 2′-5′-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates stimulator of interferon genes (STING), resulting in the recruitment of TBK1, the activation of the transcription factor IRF3 and the trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I) (Li et al., 2013; Sun, Wu, Du, Chen, & Chen, 2013; Wu et al., 2013). Another key factor is STING, encoded by the TMEM173 gene, a signaling molecule associated with the endoplasmic reticulum (ER) that is essential for controlling the transcription of type I IFNs and pro-inflammatory cytokines. STING is phosphorylated and forms intracellular clusters after recognizing 2′-5′-cGAMP released by cGAS (Ahn, Xia, et al., 2014; Ishikawa, Ma, & Barber, 2009). The regulated activation of the cGAS-STING-mediated cytosolic DNA sensing pathway is essential for cellular homeostasis, whereas erroneous signaling leads to disease phenotypes.

2. DNA metabolic factors negatively regulate immune signaling

DDR factors have long been thought to mainly sense and repair DSBs and to transmit DNA damage signaling to regulate genome integrity and cell fates. However, recent evidence suggests that the regulated functioning of these DDR factors is also critical for suppressing DNA sensing pathway-mediated immune signaling. Though defects in many DDR factors augment immune signaling, we specifically outline the contributions of NBS1, ATM, RAD51, BRCA proteins, Fanconi anemia pathway factors, DNA-PK, MRE11, MUS81 and TREX1 to immune signaling (Fig. 1).

Fig. 1.

Schematics show different stages of non-homologous end-joining, homologous recombination, base damage and replication stress processing pathways and the corresponding factors involved during various steps of these DNA metabolic pathways. Defects associated with any one of these factors will cause aging, immune disorder (immune) or cancer. FA, Fanconi anemia pathway factors; WRN, Werner syndrome protein; Polβ, DNA polymerase beta.

2.1. Role of NBS1 in innate immune signaling

NBS1 senses DNA double-strand breaks (DSBs) and plays critical roles in DSB repair, DNA damage response signaling and genome stability maintenance. The human NBS1 protein consists of 754 amino acids and contains functional domains at the N-terminus (1–196 amino acids) and the C-terminus (665–693 amino acids). The N-terminal sequence consists of a forkhead-associated (FHA) domain (20–108 amino acids) and a BRCA1 C-terminus (BRCT1: 111–197 amino acids; BRCT2: 219–327 amino acids) domain. Both the FHA and the BRCT1/2 domains directly interact with the phosphorylated H2AX (γH2AX) independently of MRE11 and BRCA1 (Kobayashi et al., 2002). Similarly, both domains interact with the phosphorylated mediator of DNA damage checkpoint 1 (MDC1), which interaction is important for stabilizing NBS1 at DSB sites (Chapman & Jackson, 2008; Melander et al., 2008). The C-terminus of NBS1 contains an MRE11-binding region (682–693 amino acids) (Tauchi et al., 2001) and an ATM-binding region (734–754 amino acids) (Bakkenist & Kastan, 2003). The NBS1-ATM interaction results in the phosphorylation of NBS1 at serine residues 278 and 343 in response to DSBs both in vitro and in vivo (Lim et al., 2000; Wu et al., 2000). NBS1 is the protein defective in Nijmegen breakage syndrome (NBS, a chromosomal instability syndrome), which is associated with immunodeficiency, microcephaly, growth retardation, a high frequency of lymphoid malignancies and an aging phenotype (Tauchi, Matsuura, Kobayashi, Sakamoto, & Komatsu, 2002). Thus, NBS1 functions as a DNA damage sensor and signal transducer in response to DNA damage to prevent genomic instability.

Recent studies show that NBS1 is also involved in immune signaling. Mice with hypomorphic Nbs1 alleles exhibit an impaired inflammatory response (Pereira-Lopes et al., 2015). Similarly, another study found that loss of NBS1 leads to exacerbated inflammation (Prochazkova et al., 2015). Furthermore, our current findings reveal that NBS1 regulates inflammatory signaling by counteracting cGAS binding to cytosolic chromatin fragments (unpublished work). NBS1 lacks any known enzymatic activities, but its physical presence on the cytosolic chromatin fragments suffices to suppress the cGAS-mediated cytosolic DNA sensing pathway. However, additional experimental evidence is required to precisely identify how NBS1 counters cGAS binding to cytosolic chromatin fragments.

2.2. Role of ATM in innate immune signaling

Ataxia telangiectasia-mutated (ATM) kinase plays a central role in mediating signal transduction in response to DSBs caused by both endogenous and exogenous genotoxic stress. ATM is present as dimers or oligomers in undamaged cells, and it is characterized by the presence of kinase domain flanked by FAT (named due to homology in this region between FRAP, ATM and TRRAP) and FATC (FAT at the extreme C-terminus) domains at the carboxyl terminal and alpha-helical HEAT (huntingtin, elongation factor 3, PP2A and TOR1) repeats at the amino terminal. Acetyltransferase Tip60 activity at the carboxyl terminal FATC domain leads to ATM activation. Activated ATM turns into monomers by auto-phosphorylation of Ser1981 in the FAT domain by the kinase domain, resulting in dissociation of its multimeric structure (Bakkenist & Kastan, 2003; Marechal & Zou, 2013). DSB-activated ATM affects phosphorylation of several substrates, such as MDC1, BRCA1, CHK2 and p53, which mediates events such as DNA repair, apoptosis, cell cycle checkpoint and other processes associated with DDR.

In humans, loss of functional ATM results in ataxia-telangiectasia (AT), a complex cancer-prone neurodegenerative disease that displays a variety of inflammatory and autoimmune syndromes, including abnormal T and B lymphocyte counts and deficient antibody responses (McKinnon, 2012). ATM functions in DNA repair during immunoglobulin and T cell receptor gene rearrangements (Bredemeyer et al., 2006). Hartlova et al. (2015) found that DNA lesions accruing from loss of ATM cause genomic DNA fragments to leak into the cytoplasm, where they activate innate immune sensors. This results in an enhanced innate resistance to viral infection and a hyper-responsiveness to a variety of innate immune stimuli (Hartlova et al., 2015). Furthermore, individuals with AT are susceptible to respiratory bacterial and chronic herpes virus infections due to defects in the adaptive immune system (Erttmann et al., 2016). Thus, ATM is critical for optimal inflammasome-dependent antibacterial innate immunity.

2.3. Role of RAD51 in innate immune signaling

RAD51, a multifunctional protein, plays central roles in DSB repair and replication fork processing. RAD51 catalyzes the core reactions of homologous recombination (HR) (Godin, Sullivan, & Bernstein, 2016). RAD51 expresses in proliferating cells, with the highest expression in the S/G₂ phase of the cell cycle (Chen et al., 1997; Godin et al., 2016; Xia, Shammas, & Shmookler Reis, 1997; Yamamoto et al., 1996). Apart from DSB repair functions, RAD51 is also essential for replication fork restart when a replication fork encounters DNA damage (Petermann, Orta, Issaeva, Schultz, & Helleday, 2010), and it prevents the accumulation of replication-associated DSBs (Lundin et al., 2003) and functions to maintain genome stability. Germ-line mutations in the RAD51 gene lead to embryonic death (Tsuzuki et al., 1996).

Evidence on the role of RAD51 in immune signaling is very limited. A study by Wolf et al. (2016) indicated that RAD51 prevents the release of short nuclear DNA fragments into the cytosol by binding to these DNA fragments, thereby preventing immune response signaling (Wolf et al., 2016). Bhattacharya et al. (2017) have identified a role for RAD51 in innate immune signaling in response to DNA damage and replication stress caused by radiation (Bhattacharya et al., 2017). According to their findings, RAD51 is recruited to the sites of perturbed replication forks and DSBs, which blocks the excess exonuclease activity of MRE11 on the newly replicated genome and on DSB repair, respectively. Consequently, this limits the accumulation of self-DNA in the cytosol and prevents the initiation of STING-mediated innate immune signaling. Thus, RAD51’s coordinated activities in DSB repair and replication fork maintenance suppress innate immune signaling.

2.4. Role of BRCA1 in immune signaling

BRCA1 is a multifunctional protein that physically interacts both directly and indirectly with specific partner proteins (Huen, Sy, & Chen, 2010). BRCA1 is critical for maintaining genome stability largely because of its role in HR-mediated DSB repair and replication fork stability. In the absence of BRCA1, cells repair DSBs by NHEJ, resulting in increased chromosomal instability and genomic alterations (Venkitaraman, 2014). Similarly, BRCA1 deficiency interferes with RAD51 nucleofilament formation, causing MRE11-mediated degradation of newly replicated genome (Schlacher et al., 2011; Schlacher, Wu, & Jasin, 2012; Su et al., 2014). Moreover, BRCA1 regulates key effectors that control the G₂/M cell cycle checkpoint and is involved in regulating the onset of mitosis (Yarden, Pardo-Reoyo, Sgagias, Cowan, & Brody, 2002). Breast tumors that arise in BRCA1 mutation carriers typically manifest as high-grade basal-like breast cancers (Berry, Parmigiani, Sanchez, Schildkraut, & Winer, 1997; Couch et al., 1997; Foulkes, Smith, & Reis-Filho, 2010; Roy, Chun, & Powell, 2011). Despite these well-characterized functions of BRCA1 in DSB repair, replication fork instability and the DDR signaling, the molecular events responsible for triggering breast carcinogenesis in carriers of BRCA1 mutations remains undetermined.

BRCA1’s involvement in suppressing immune signaling is very limited. Reports suggest that BRCA1-mutated breast tumors harbor a significantly higher number of CD3positive and CD8positive tumor infiltrating lymphocytes (TILs), as well as higher expression of PD-1 and PD-L1 in tumor-associated immune cells, than BRCA1-proficient tumors (Lakhani et al., 1998; Strickland et al., 2016). Additionally, BRCA1 plays a hitherto unidentified role as a cofactor to IFI16 in the nuclear innate sensing of foreign DNA and subsequent assembly and cytoplasmic distribution of stable IFI16-inflammasomes leading into IL-1β formation, as well as the induction of IFN-β via cytoplasmic signaling through IFI16, STING, TBK1 and IRF3 (Dutta et al., 2015). Another study has identified activation of STING-mediated chemokine production in response to endogenous or exogenous S-phase specific DNA damage in vitro, resulting in an inflammatory microenvironment in BRCA1-mutant breast tumors (Parkes et al., 2017). However, the initial events responsible for triggering immune signaling in BRCA1-mutant tumors remain to be identified.

2.5. Role of Fanconi anemia pathway factors in immune signaling

Fanconi anemia (FA) is a genetically heterogeneous disease, with 16 complementation groups identified to date (Crossan & Patel, 2012; Kee & D’Andrea, 2010; Kottemann & Smogorzewska, 2013; Moldovan & D’Andrea, 2009). A central event in the FA pathway is the mono-ubiquitination of FANCD2 and FANCI upon DNA damage. This modification is mediated by the FA core complex, which consists of eight proteins (FANCA, B, C, E, F, G, L and M) (Garcia-Higuera et al., 2001; Smogorzewska et al., 2007). Upon ubiquitination, FANCD2 and FANCI heterodimerize and functionally interact with downstream FA proteins such as FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, FANCP/SLX4 and RAD51C, and their associated protein, BRCA1 (Bunting & Nussenzweig, 2010; Crossan & Patel, 2012; Kee & D’Andrea, 2010; Moldovan & D’Andrea, 2009). FA factors play roles in replication, recombination, repair and recovery in response to a variety of DNA damaging agents. Zhu et al. (2015) found that the FA pathway is critical for repairing clustered DSBs (DSBs closely associate with single strand breaks and base lesions) specifically in the S/G₂ phase of the cell cycle, mainly because of defective RPA2 and RAD51 recruitment to the sites of DSBs (Zhu et al., 2015). In addition, replication fork processing is also defective in the absence of FANCD2. Consequently, FANCD2-deficient cells exhibit high levels of anaphase bridge formation and defective chromosome segregation. Additionally, Schlacher et al. (2012) found that the FA pathway is critical for blocking MRE11-mediated degradation of replication forks (Schlacher et al., 2012).

FA is a rare inherited syndrome that is characterized by congenital abnormalities, progressive bone marrow failure and a highly elevated risk of hematological and squamous cell cancer (Fanconi, 1967). In vitro stimulated FA bone marrow showed elevated levels of tumor necrosis factor-alpha (TNF-α) and IFN-γ (Dufour et al., 2003). Recent evidence suggests that FA genes function in the selective autophagy of genetically distinct viruses, in mitochondrial quality control and in preventing inflammasome activation due to mitochondrial reactive oxygen species (ROS) (Sumpter et al., 2016). However, whether chromosome mis-segregation together with replication-fork degradation initiates immune signaling in the absence of a functional FA protein remains to be evaluated.

2.6. Role of DNA-PK in immune signaling

DNA-PKcs is a member of the PIKK family of protein kinases and is abundantly expressed in almost all mammalian cells (Hartley et al., 1995). It forms a complex with KU70 and KU80 (also called KU86), which are encoded in humans by the XRCC6 and XRCC5 genes, respectively, have a strong affinity for free ends of DNA (Blier, Griffith, Craft, & Hardin, 1993) and help DNA-PKcs to bind to DSBs (Uematsu et al., 2007). DNA-PKcs, a 469-kDa protein encoded by the PRKDC gene in humans, is composed of several distinct functional domains: a highly conserved catalytic kinase domain; a large N-terminal domain containing mostly helical elements and HEAT repeats, along with the JK, PQR and ABCDE phosphorylation clusters; and the FAT and FATC domains, both of which are conserved among PIKK family members (Dobbs, Tainer, & Lees-Miller, 2010). The FAT and FATC domains surround the catalytic domain and serve to stabilize conformational changes to the catalytic core and regulate kinase activity (Rivera-Calzada, Maman, Spagnolo, Pearl, & Llorca, 2005). DNA-PK complex is essential for the NHEJ-mediated repair of DSBs in all cell cycle phases. In the absence of this complex, cells utilize an alternative-NHEJ mechanism, causing genomic instability (Neal & Meek, 2011). Besides its function in DSB repair, DNA-PKcs also phosphorylates a wide variety of proteins that function in various cellular processes.

Germline mutations targeting DNA-PKcs lead to severe combined immunodeficiency (SCID) (van der Burg et al., 2009). DNA-PK complex plays critical functions in the immune system. DNA-PKcs is required for V(D)J recombination, which utilizes the NHEJ pathway to promote antigen diversity in the mammalian immune system. DNA-PKcs interacts with DNA in the cytoplasm and is critical for inducing innate immune responses to DNA and DNA viruses in fibroblasts (Ferguson, Mansur, Peters, Ren, & Smith, 2012; Monroe et al., 2014; Zhang et al., 2011). DNA-PK is critical for NF-κB-mediated expression of VCAM-1 in response to treatment with the cytokine tumor necrosis factor (TNF) (Ju et al., 2010). More recent studies have revealed a novel role for DNA-PK in activating innate immunity and inflammation response independently of NF-κB, as they found that DNA from various pathogens is bound by DNA-PK, resulting in IRF-3-mediated transcription of multiple cytokine and chemokine genes independently of DNA-PKcs kinase activity (Ferguson et al., 2012). Furthermore, evidence shows that KU70 induces IFN-λ1 production by activating IFN regulatory factor (IRF)-1 and IRF-7 in response to cytosolic DNA (Nakad & Schumacher, 2016; Zhang et al., 2011). Thus, DNA-PK has a limited, yet an important role in activating immune signaling.

2.7. Role of meiotic recombination 11 homolog A (MRE11) in immune signaling

MRE11 is a highly conserved 70—90kDa protein with an N-terminal Mn²⁺/Mg²⁺-dependent phosphoesterase domain and two distinct C-terminal DNA binding domains that possess various biochemical activities, such as intrinsic DNA binding activity, with the specific ability to synapse DSB termini. It also performs endo- and exo-nuclease activities against a variety of ssDNA and dsDNA (double-strand DNA) substrates (de Jager et al., 2001; Hopkins & Paull, 2008; Usui et al., 1998; Williams et al., 2008). Initiating the DDR as part of the MRN complex, MRE11 participates in nuclease activities involved in both NHEJ and HR. Shibata et al. designed a structure-based exo- and endonuclease activity inhibitor for MRE11 and showed that MRE11’s exonuclease activity alone suffices to ensure DNA damage repair by the NHEJ pathway, whereas MRE11’s endonuclease activity and exonuclease activity are both necessary for DSB repair by HR (Shibata et al., 2014). Because it cannot perform the 5′→3λ′ exonuclease activity required to have 3′ ssDNA overhangs for HR, MRE11 acts in association with other factors and proteins that generate 3′ overhangs (Hopkins & Paull, 2008; Mimitou & Symington, 2009). Mutation in MRE11 causes ataxia telangiectasia-like disorder (ATLD). The clinical features of patients with ATLD are very similar to those with AT, but ATLD is characterized by a later onset of the neurological features and slower progression of the disorder in the early years. ATLD patients also show normal levels of total IgG, IgA and IgM, though there may be reduced levels of specific functional antibodies (Hernandez et al., 1993).

Evidence suggests both direct and indirect roles for MRE11 in immune signaling. MRE11 is known to function at perturbed replication forks. Evidence shows that lack of BRCA1/2, FA factors, RAD51, WRN and XPG1 leads to MRE11-mediated excessive processing of newly replicated genome. A recent report strongly suggests that lack of RAD51 leads to MRE11-mediated degradation of newly replicated genome and the accumulation of these fragmented self-DNA in the cytoplasm, culminating in the activation of STING-mediated innate immune signaling (Bhattacharya et al., 2017). In addition to this indirect mechanism, MRE11 physically senses exogenous dsDNA in the cytoplasm, resulting in the activation of STING and IRF3 (Kondo et al., 2013). Thus, MRE11 plays important roles not only in directly recognizing exogenous dsDNA but also in releasing self-DNA into the cytoplasm, resulting in the initiation of STING-dependent signaling, in addition to its role in DNA damage responses.

2.8. Role of MMS and UV-sensitive protein 81 (MUS81) in immune signaling

The XPF/MUS81 endonuclease family plays a role in resolving recombination intermediates during DNA repair after inter-strand cross-links, replication fork collapse and DSBs. The encoded protein associates with one of two closely related essential meiotic endonuclease proteins (EME1 or EME2) to form a complex that processes DNA secondary structures. This complex contains an N-terminal DEAH domain, an excision repair cross complementation group 4 (ERCC4) endonuclease domain and two tandem C-terminal helix-hairpin-helix domains. Mice with a homozygous knockout of the orthologous gene have significant meiotic defects, including the failure to repair a subset of DSBs. MUS81 suppresses chromosomal instability arising from stalled replication forks by cleaving to potentially detrimental DNA structures (Ciccia, McDonald, & West, 2008).

Ho et al. (2016) found that, like MRE11, MUS81 causes the accumulation of fragmented self-DNA, including non-B DNA structures, repetitive sequences, DNA lesions, R-loops and common fragile sites, as a result of replication stress. The resultant fragmented DNA leads to STING-dependent expression of type I IFNs and chemokines (Ho et al., 2016). The direct role of MUS81 in immune signaling is not well understood. Future experimental evidence is required to completely understand whether abnormalities in genomic stability associated with MUS81 defects contribute to immune signaling.

2.9. Role of three prime repair exonuclease 1 (TREX1) in immune signaling

TREX1 is the major exonuclease in mammalian cytoplasm, and it acts on both ssDNA (Hoss et al., 1999; Mazur & Perrino, 1999) and dsDNA (Grieves et al., 2015). TREX1 was originally thought to be a proofreading enzyme for DNA polymerases α and β, which lack intrinsic 3′ exonuclease activities. It has recently been discovered that the 3′→5′ DNA exonucleolytic function of TREX1 is essential for clearing DNA from the cytoplasm (Stetson, Ko, Heidmann, & Medzhitov, 2008; Yang, Lindahl, & Barnes, 2007). DNA accumulation in TREX1-deficient cells due to viral infection or as a byproduct of DNA repair stimulates an immune response (Stetson et al., 2008; Yang et al., 2007).

The major function of TREX1 is to maintain the host’s innate immune tolerance to self-DNA. TREX1 degrades dsDNA, preventing this polynucleotide from acting as an autoantigen to inappropriately activate the immune system (Reina-San-Martin, Nussenzweig, Nussenzweig, & Difilippantonio, 2005; Stetson et al., 2008). Mutations in human TREX1 cause a spectrum of autoimmune disorders, including Aicardi–Goutieres syndrome, familial chilblain lupus and retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus (DiFrancesco et al., 2015; Lee-Kirsch, Wolf, & Gunther, 2014; Rice et al., 2007). TREX1-deficient mice exhibit profound systemic inflammation affecting multiple organs, elevated autoantibody production and inflammatory myocarditis early in age (Morita et al., 2004; Stetson et al., 2008). Mutations that disrupt the mouse’s TREX1 DNase activity lead to the accumulation of self-DNA in the cytosol, which activates the cGAS-STING-mediated type I IFN response and systemic inflammation. The inflammation and mortality of TREX1-null mice can be genetically rescued by depleting cGas^−/−, sting^−/−, ifnar1^−/−, or irf3^−/− (Ahn, Ruiz, & Barber, 2014; Gall et al., 2012; Gao et al., 2015; Gray, Treuting, Woodward, & Stetson, 2015; Stetson et al., 2008). Additionally, pharmacological inhibition of TBK1, a key serine/threonine kinase that phosphorylates STING and IRF3, alleviates autoimmune disease phenotypes and increases overall survival of TREX1^−/− mice (Hasan et al., 2015). Thus, the nuclease activity of TREX1 helps cells to clear the self- and foreign DNA in the cytosol, thereby preventing unwarranted initiation of autoimmune signaling.

3. Mechanisms of immune signaling regulation by DDR factors

Immune signaling is triggered by the presence of DNA in unusual locations, such as the cytoplasm or the endosomes, as DNA is normally located in the nucleus of eukaryotic cells (Ishii & Akira, 2006; Kerur et al., 2011; Lund et al., 2003; Stetson et al., 2008). The DNA can be foreign DNA, like viral infections or bacterial infections, or it can be the organism’s self-DNA generated by both endogenous and exogenous genotoxic stress. As described before, DNA repair, sensing and signaling factors are critical for the timely repair of DNA lesions, replication fork stability and cell cycle progression. By participating in these DNA metabolic activities, DDR factors prevent unwarranted activation of immune signaling. Below are some of the major mechanisms by which DDR factors contribute to the accumulation, sensing and removal of cytosolic DNA, as well as the activation and suppression of immune signaling (Fig. 2).

Fig. 2.

Schematics show the mechanism of initiation and maintenance of immune signaling in cells defective for DNA sensing, repairing and signaling factors (DDR). Excessive processing of newly replicated genome by nucleases, normal DNA replication and DNA repair or defective G₂/M checkpoint followed by cytokinesis in cells defective in DDR factor result in the accumulation of self-DNA in the cytosol, fragmented DNA in the nucleus transported to cytosol and accumulation of chromatin fragments in the cytosol, respectively. Subsequently, cytosolic DNA sensing cGAS-STING-TBK1-IRF3 pathway initiates expression of immune genes, culminating in the establishment of aging, immune disorder or cancer.

3.1. Generation of chromatin fragments due to defective G₂/M checkpoint

G₂/M checkpoint arrest is rapidly activated in response to DNA damage, which results in the timely processing of damaged DNA before mitosis. However, if release from G₂ arrest occurs before the DNA damage repair is complete or progression of cells with fused chromosome from G₂ to mitotic phase, micronuclei will form upon cytokinesis. Recent findings have provided mechanistic insights into how the accumulation of chromatin fragments in the form of micronuclei and the subsequent rupture of the nuclear envelope initiate the cGAS-STING-mediated cytosolic DNA sensing pathway (Bartsch et al., 2017; Dou et al., 2017; Gluck et al., 2017; Harding et al., 2017; Mackenzie et al., 2017; Yang, Wang, Ren, Chen, & Chen, 2017). It is well known that deficiencies in DDR factors, including ATM and BRCA1/2, with known functions in cell cycle checkpoint activation in response to genotoxic stress lead to chromatin fragment accumulation in the cytoplasm. Therefore, defective DSB repair together with checkpoint releases before the completion of DNA repair contributes to the formation of micronuclei and the activation of the cGAS-mediated cytosolic DNA sensing pathway.

3.2. Degradation of replication forks by nucleases

Replication fork instability due to nuclease-mediated degradation of newly replicated genomic DNA can contribute to self-DNA accumulation in the cytoplasm. Recent evidence suggests that defects in BRCA1/2, FANCA and WRN lead to MRE11-mediated degradation of newly replicated genomic DNA (Schlacher et al., 2011, 2012; Su et al., 2014). An elegant study by Bhattacharya et al. (2017) showed how MRE11-mediated excessive processing of newly replicated genome in the absence of RAD51 leads to the accumulation of genomic DNA in the cytoplasm (Bhattacharya et al., 2017). Similarly, MUS81 causes the accumulation of fragmented non-B DNA structures, repetitive sequences, DNA lesions, R-loops and common fragile sites in the cytoplasm in response to replication stress (Ho et al., 2016). The accumulation of these fragmented self-DNA in the cytoplasm triggers STING-mediated innate immune signaling. Thus, defects in factors that regulate the enzymatic activities of MRE11 and MUS81 in response to genotoxic stress lead to self-DNA accumulation, resulting in the activation of the cytosolic DNA sensing pathway-mediated immune signaling pathway.

3.3. Failure to retain fragmented self-DNA within the nucleus

Cells can produce fragmented DNA within the nucleus as a byproduct of DNA replication and repair in response to both exogenous and endogenous genotoxic stress. Multiple DDR factors bind to these fragmented DNA because of their inherent non-DNA sequence-specific interaction characteristics. As a result, the DNA fragments are retained within the nuclei, preventing their export or release into the cytoplasm. For example, RPA2 and RAD51 prevent the release of short nuclear DNA fragments into the cytosol by binding to these fragments (Wolf et al., 2016). Defects in DNA binding factors that retain fragmented self-DNA within the nucleus lead to self-DNA accumulation in the cytosol, culminating in the activation of cytosolic DNA sensing pathway-mediated immune signaling.

3.4. Direct sensing of DNA in the cytoplasm

Most of the nuclear DNA damage sensors play an important part not only in detecting foreign and self-DNA in the cytoplasm but also in activating immune signaling, because of their non-sequence-specific DNA recognition characteristics. Though most DDR factors are localized in the nucleus, a small portion of these factors reside in the cytoplasm. For example, cytoplasmic MRE11 (together with its binding partner RAD50) senses dsDNA in the cytoplasm, leading to the activation of a STING-dependent type 1 IFN response (Kondo et al., 2013). KU70 recognizes cytosolic DNA and induces the production of IFN-λ1 (a member of Type-III IFN) rather than Type-I IFN. This induction is mediated via the activation of IFN regulatory factor (IRF)-1 and IRF-7 (Zhang et al., 2011). Similarly, DNA-PK binds to cytoplasmic DNA, resulting in STING-TBK1-mediated activation of type I IFN and cytokine and chemokine genes (Ferguson et al., 2012). So, DNA damage sensors can also recognize foreign and self-DNA in the cytosol and activate downstream cytosolic DNA sensing pathway-mediated immune signaling.

3.5. Removal of self-DNA from the cytoplasm

The accumulation of nuclear-derived DNA, either in the form of small DNA fragments or large chromatin fragments, is the main trigger for activating the cytosolic DNA sensing pathway. Therefore, the efficient and timely removal of DNA fragments from the cytoplasm determine the fate of cells. Currently, TREX1 is the only known cytosolic exonuclease in mammalian cytoplasm, and it acts on both ssDNA (Hoss et al., 1999; Mazur & Perrino, 1999) and dsDNA (Grieves et al., 2015). As discussed above, in the absence of TREX1, ssDNA and dsDNA derived from endogenous retroelements accumulate in the cytoplasm and cause Aicardi-Goutieres syndrome (AGS) and chilblain lupus. Thus, the dysregulation of factors involved in removing DNA from the cytoplasm can permanently activate the DNA sensing pathway.

4. Consequences of defective DDR factors mediated immune signaling

Defects in DDR factors trigger multitude of cellular phenotypes, including autoinflammatory disease, cellular senescence and cancer. Genotoxic agents serve as initial triggers for immune signaling activation which is a consequence of dysfunctional DDR factors. Below are some of the major consequences of defective DDR factors mediated immune signaling.

4.1. Cytosolic DNA sensing pathway-mediated cellular senescence

Senescence is an intrinsic cellular response that induces irreversible cell cycle arrest and plays a critical role in both aging and tumor suppression. Though the triggers for cellular senescence are manifold, the activation of DDR is thought to be the common mechanism that is critical for inducing and maintaining a senescence phenotype (Fumagalli & d’Adda di Fagagna, 2009). We can distinguish two types of cellular senescence: replicative senescence, which depends on telomere shortening, and stress-induced premature senescence, which does not depend on telomere shortening. Several studies strongly support the idea that cGAS plays an essential role in initiating the signals required for telomere shortening-independent premature senescence (Dou et al., 2017; Gluck et al., 2017; Yang et al., 2017). However, the question of how cGAS establishes the senescence phenotype remains unanswered (Li & Chen, 2018). This is intriguing, because cGAS recognition of cytosolic DNA fragments and the subsequent activation of innate immune signaling precede the appearance of the senescence phenotype, and depleting cGAS or inhibiting its activity suffices to ameliorate premature senescence in the presence of cytosolic chromatin fragments. Thus, the initial activation of the cGAS-mediated cytosolic DNA sensing pathway is sufficient to establish a senescence phenotype. Reports suggest that IL-8 and IL-6 are known to feed back to the secreting cells to reinforce senescence signaling (Acosta et al., 2008; Kuilman et al., 2008). Therefore, cGAS-mediated expression of pro-inflammatory factors, such as IFN-β and IL-6, and IL-8, in response to defective DDR signaling could serve as a paracrine signal that establishes a senescence phenotype upon DNA damage caused by genotoxic stress.

4.2. Crosstalk among defective DDR factors, immune signaling and cancer

The major function of DDR factors is to repair damaged DNA to promote genomic stability and suppress cancer. Available evidence strongly shows that cancer develops in the absence of almost all DDR factors, though the nature and the site of cancer vary. Mechanistically, lack of DDR factors leads to erroneous DNA repair, replication fork instability, cell cycle checkpoint defects and genomic instability in response to genotoxic stress. DDR-defective cells accumulate mutations in their genome, which results in the generation of neoantigens, the accumulation of fragmented DNA in the cytosol and persistent activation of DDR signaling. These outcomes feed into the immune system, which activates immune signaling. However, immune signaling is a double-edged sword. On one hand, it helps to remove cancer cells (anti-tumor); but, on the other hand, it promotes carcinogenesis (pro-tumor). Some recent studies indicate the activation of DNA cytosolic sensing pathways in cancer cells: the STING pathway in HT1080 fibrosarcoma cells after RAD51 depletion (Bhattacharya et al., 2017); the cGAS-STING signaling pathway in BRCA1-mutant breast cancer cells (Parkes et al., 2017); and cytosolic DNA response in a variety of metastatic cancers (Bakhoum et al., 2018). However, it is unclear how this intrinsic immune signaling activation characteristic of DDR defects promotes tumorigenesis. In contrast, other recent evidence shows that the cytosolic DNA sensing pathway acts as an intrinsic barrier to tumorigenesis. For example, MUS81-dependent accumulation of genomic DNA in the cytoplasm not only triggers STING-mediated type 1 IFNs but also rejects prostate cancer (Wolf et al., 2016). Ionizing radiation-induced DNA damage in B16 cells serves as a vaccine that promotes STING-dependent tumor rejection in combination with immune checkpoint blockage (Harding et al., 2017). A detailed understanding of the factors that determine whether the immune system eliminates cancer cells or fuels tumorigenesis will help in developing novel therapeutic approaches to both prevent and treat cancers.

4.3. Interconnection between defective DDR factors and autoinflammatory disease

Self-DNA has long been considered a key cause of autoinflammatory disease. Based on the recent insights into the innate immune pathways and sensors that instigate self-DNA, there are two major pathways responsible for the development of autoinflammatory disease: (a) aberrant activation of the cytosolic DNA sensing pathway due to defective DDR factors and (b) loss- or gain-of-function mutations in cytosolic DNA degradation enzymes and the cytosolic DNA sensing pathway. For example, ATM deficiency leads to autoinflammation and elevated levels of circulating type 1 IFN in AT patients, as a result of dysfunctional V(D)J recombination (Siddoo-Atwal, Haas, & Rosin, 1996; Sugihara, Murano, Nakamura, Ichinohe, & Tanaka, 2011). Intriguingly, depleting either cGAS or STING attenuated autoinflammatory disease in ATM-deficient mice (Ahn, Xia, et al., 2014; Hartlova et al., 2015; Lan, Londono, Bouley, Rooney, & Hacohen, 2014). Mutations in genes involved in nucleic acid metabolism, including TREX1, rnaseH2a, rnaseH2b and samhd1, cause AGS. Evidence indicates that, similar to human AGS patients with TREX1 deficiency, trex1^−/− mice also develop an inflammatory phenotype (Crow et al., 2006). Importantly, genetic depletion of cGAS or STING in trex1^−/− mice rescues autoinflammatory symptoms associated with trex1 deficiency (Ahn, Ruiz, et al., 2014; Gall et al., 2012; Gao et al., 2015; Gray et al., 2015). Thus, constitutive activation of the cytosolic DNA sensing pathway due to defective DNA damage sensors and nuclease factors contributes to the development of autoinflammatory disease.

5. Conclusions

Most of the research on DNA repair machinery focuses mainly on its functions in DNA damage sensing, repair and signaling. All of these activities are essential for cellular homeostasis. Defects in DDR machinery lead to a multitude of deleterious effects, including immune disorder, aging and cancer. However, components of the DDR machinery clearly play roles in suppressing unwarranted immune signaling as well. A more precise understanding of these roles can be applied to suppress erroneous immune signaling due to defects in DDR factors or exploited to redirect immune signaling to eliminate cancer and senescent cells.

Abbreviations

ATM

ataxia telangiectasia-mutated

ATR

ATM and Rad-3-related

cGAS

cyclic GMP-AMP synthase

DDR

DNA damage response

DNA-PK

DNA-dependent protein kinase

DNA-PKcs

catalytic subunit of DNA-PK

DSB

double-strand breaks

dsDNA

double-strand DNA

FA

Fanconi anemia

HR

homologous recombination

IFI16

interferon (IFN)-inducible gene 16

IFN-I

interferon-I

IRF3

interferon regulatory factor 3

MRN

Mre11 nuclease, Rad50 coiled-coil protein, and Nbs1 regulator protein

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NHEJ

non-homologous end-joining

PD-1

Programmed cell death protein 1

PRRs

pattern recognition receptors

ROS

reactive oxygen species

RPA

replication protein

ssDNA

single-strand DNA

STING

stimulator of IFN genes

TBK1

TANK-binding kinase 1

TILs

tumor infiltrating lymphocytes

TREX1

Three prime repair exonuclease 1