Genome instability syndromes caused by impaired DNA repair and aberrant DNA damage responses

Maintenance of genome integrity is essential for all organisms because genome information regulates cell proliferation, growth arrest, and vital metabolic processes in cells, tissues, organs, and organisms. Because genomes are constantly exposed to intrinsic and extrinsic genotoxic stress, cellular DNA repair machinery and proper DNA damage responses (DDR) have evolved to quickly eliminate genotoxic DNA lesions, thus maintaining the genome integrity suitably. In human, germline mutations in genes involved not only in cellular DNA repair pathways but also in cellular DDR machinery frequently predispose hereditary diseases associated with chromosome aberrations. These genetic syndromes typically displaying mutations in DNA repair/DDR-related genes are often called “genome instability syndromes.” Common features of these hereditary syndromes include a high incidence of cancers and developmental abnormalities including short stature, microcephaly, and/or neurological deficiencies. However, precisely how impaired DNA repair and/or dysfunctional DDR pathologically promote(s) these syndromes are poorly understood. In this review article, we summarize the clinical symptoms of several representatives “genome instability syndromes” and propose the plausible pathogenesis thereof.     

Genistein ameliorates cardiac inflammation and oxidative stress in streptozotocin-induced diabetic cardiomyopathy in rats

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)—a highly cytotoxic DNA lesion—activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.     

Role of deubiquitinases in DNA damage response

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.     

Stable Cellular Senescence Is Associated with Persistent DDR Activation

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.     

The multi-functionality of UHRF1: epigenome maintenance and preservation of genome integrity

During S phase, the cooperation between the macromolecular complexes regulating DNA synthesis, epigenetic information maintenance and DNA repair is advantageous for cells, as they can rapidly detect DNA damage and initiate the DNA damage response (DDR). UHRF1 is a fundamental epigenetic regulator; its ability to coordinate DNA methylation and histone code is unique across proteomes of different species. Recently, UHRF1’s role in DNA damage repair has been explored and recognized to be as important as its role in maintaining the epigenome. UHRF1 is a sensor for interstrand crosslinks and a determinant for the switch towards homologous recombination in the repair of double-strand breaks; its loss results in enhanced sensitivity to DNA damage. These functions are finely regulated by specific post-translational modifications and are mediated by the SRA domain, which binds to damaged DNA, and the RING domain. Here, we review recent studies on the role of UHRF1 in DDR focusing on how it recognizes DNA damage and cooperates with other proteins in its repair. We then discuss how UHRF1’s epigenetic abilities in reading and writing histone modifications, or its interactions with ncRNAs, could interlace with its role in DDR.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.Key words: genomic stability, DNA repair, double-strand breaks, ATM, proteasome, PA28γ (PSME3)     

Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair.     

The DNA damage response: ten years after

The DNA damage response (DDR), through the action of sensors, transducers, and effectors, orchestrates the appropriate repair of DNA damage and resolution of DNA replication problems, coordinating these processes with ongoing cellular physiology. In the past decade, we have witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage. These findings have important implications for aging and cancer.     

DNA damage during meiosis induces chromatin remodeling and synaptonemal complex disassembly

DNA damage to the germline genome must be accurately repaired to ensure transmission of intact genetic information to following generations. Meiosis presents challenges to the DNA damage response (DDR) because it universally requires changes to chromosome structure that can affect DNA repair outcomes. We report the existence of a meiotic DDR at chromosome axes that results in chromatin remodeling, synaptonemal complex disassembly, and axis separation in response to irradiation at late pachytene stages in C. elegans. The axis component HTP-3 is required for germline acquisition of H2AacK5, an axis-specific chromatin mark that is DNA damage responsive. Irradiated wild-types show reduction of H2AacK5 and axis separation that are dependent on the acetyltransferase MYS-1/TIP60. Restoration of H2AacK5 levels requires ATM-1 kinase and correlates with resynapsis. We propose that the meiotic DDR involves early chromatin remodeling at chromosome axes to dismantle structures promoting interhomolog recombination and facilitate efficient nonhomolog-based repair before pachytene exit.     

miR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated (ATM) kinase

The DNA damage response (DDR) encompasses multi-step processes by which cells evolve to sense DNA damage, transduce the signal and initiate the repair of damaged DNA. Ataxia Telangiectasia Mutated (ATM) Kinase, which functions as the primary sensor and transducer of DNA damage signal, has been demonstrated to play an important role in the DDR and cancer prevention. Hence, understanding the molecular mechanisms underlying the regulation of ATM has received much attention. Here, we found that miR-18a was upregulated in both cell lines and patients' tissue samples of breast cancer. Furthermore, we demonstrated that ectopically expressing miR-18a downregulated ATM expression by directly targeting the ATM-3'-UTR and abrogated the IR-induced cell cycle arrest. Similar to the effect of ATM siRNA, overexpressing miR-18a in breast cancer cells reduced the DNA damage repair ability and the efficiency of homologous recombination-based DNA repair (HRR) and sensitized cells to γ-irradiation (IR) treatment. However, inhibition of miR-18a led to augmentation of DNA damage repair, increase of HRR efficiency and reduced cellular radiosensitivity. Moreover, we showed that the phorsphorylation level and nuclear foci formation of H2AX and 53BP1, the downstream substrates of ATM kinase, were significantly deceased in miR-18a overexpressing cells. Taken together, our results uncover a new regulatory mechanism of ATM expression and suggest that miR-18a might be a novel therapeutic target.     

Listeria monocytogenes Dampens the DNA Damage Response

The DNA damage response (DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O (LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

ZNF212 promotes genomic integrity through direct interaction with TRAIP

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.     

Hyperactivation of DNA-PK by Double-Strand Break Mimicking Molecules Disorganizes DNA Damage Response

Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an “all-or-none” pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a “false” DNA damage signaling that disorganizes the DNA repair system.     

DNA-damage checkpoints: location, location, location

The DNA-damage response (DDR) is an evolutionarily conserved signaling cascade crucial for sensing DNA damage and activating cellular responses such as cell-cycle arrest, DNA repair, senescence and apoptosis. Excitingly, two recent studies describe activation of this checkpoint in the absence of DNA damage. These studies support the idea that accumulation of checkpoint proteins and changes in global-chromatin structure are important signaling intermediates for the activation of the DDR.