The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle

Human genomic instability syndromes affect the nervous system to different degrees of severity, attesting to the vulnerability of the CNS to perturbations of genomic integrity and the DNA damage response (DDR). Ataxia-telangiectasia (A-T) is a typical genomic instability syndrome whose major characteristic is progressive neuronal degeneration but is also associated with immunodeficiency, cancer predisposition and acute sensitivity to ionizing radiation and radiomimetic chemicals. A-T is caused by loss or inactivation of the ATM protein kinase, which mobilizes the complex, multi-branched cellular response to double strand breaks in the DNA by phosphorylating numerous DDR players. The link between ATM's function in the DDR and the neuronal demise in A-T has been questioned in the past. However, recent studies of the ATM-mediated DDR in neurons suggest that the neurological phenotype in A-T is indeed caused by deficiency in this function, similar to other features of the disease. Still, major issues concerning this phenotype remain open, including the presumed differences between the DDR in post-mitotic neurons and proliferating cells, the nature of the damage that accumulates in the DNA of ATM-deficient neurons under normal life conditions, the mode of death of ATM-deficient neurons, and the lack of a major neuronal phenotype in the mouse model of A-T. A-T remains a prototype disease for the study of the DDR's role in CNS development and maintenance.     

DNA damage accumulation and repair defects in acute myeloid leukemia: implications for pathogenesis,disease progression,and chemotherapy resistance

DNA damage repair mechanisms are vital to maintain genomic integrity. Mutations in genes involved in the DNA damage response (DDR) can increase the risk of developing cancer. In recent years, a variety of polymorphisms in DDR genes have been associated with increased risk of developing acute myeloid leukemia (AML) or of disease relapse. Moreover, a growing body of literature has indicated that epigenetic silencing of DDR genes could contribute to the leukemogenic process. In addition, a variety of AML oncogenes have been shown to induce replication and oxidative stress leading to accumulation of DNA damage, which affects the balance between proliferation and differentiation. Conversely, upregulation of DDR genes can provide AML cells with escape mechanisms to the DDR anticancer barrier and induce chemotherapy resistance. The current review summarizes the DDR pathways in the context of AML and describes how aberrant DNA damage response can affect AML pathogenesis, disease progression, and resistance to standard chemotherapy, and how defects in DDR pathways may provide a new avenue for personalized therapeutic strategies in AML.     

Immunological disorders and DNA repair

Cellular DNA continuously incurs damage and a range of damage response mechanisms function to maintain genomic integrity in the face of this onslaught. During the development of the immune response, the cell utilises three defined processes, V(D)J recombination, class switch recombination and somatic hypermutation, to create genetic diversity in developing T and B cells. Curiously, the damage response mechanisms employed to maintain genomic stability in somatic cells have been exploited and adapted to help generate diversity during immune development. As a consequence of this overlap, there is mounting evidence that disorders attributable to impaired damage response mechanisms display associated immunodeficiency. Since double strand breaks (DSB) are created during at least two of the mechanisms used to create immunoglobulin diversity, namely V(D)J recombination and class switch recombination, it is not surprising that disorders associated with defects in the response to double strand breaks are those most associated with immunodeficiency. Here, we review the steps involved in the generation of genetic diversity during immune development with a focus on the damage response mechanisms employed and then consider human immunodeficiency disorders associated with impaired damage response mechanisms.     

Human DNA damage response and repair deficiency syndromes: Linking genomic instability and cell cycle checkpoint proficiency

A plethora of clinically distinct human disorders exist whose underlying cause is a defect in the response to or repair of DNA damage. The clinical spectrum of these conditions provides evidence for the role of the DNA damage response (DDR) in mediating diverse processes such as genomic stability, immune system function and normal human development. Cell lines from these disorders provide a valuable resource to help dissect the consequences of compromised DDR at the molecular level. Here we will discuss some well known, less well known and ‘novel’ DDR defective disorders with particular reference to the functional interplay between the DNA damage response and cell cycle checkpoints. We will describe recent advances in further delineating the genetic basis of Seckel syndrome and microcephalic osteodysplastic primordial dwarfism type II, which have shed more light on the interplay between the DDR, cycle progression and centrosomes. We will also overview recent developments concerning haploinsufficiency of DDR components and their association with certain genomic disorders such as Miller–Dieker lissencephaly syndrome and Williams–Beuren syndrome. Finally, we will discuss how defects in the DDR result in some unexpected clinical features before describing how the nature of a DDR defect impacts on the management and treatment of individuals with these conditions.     

SUMOylation and de-SUMOylation in response to DNA damage

To maintain genomic integrity, a cell must utilize multiple mechanisms to protect its DNA from the damage generated by environmental agents or DNA metabolism. SUMO (small ubiquitin-like modifier) can regulate protein stability, protein cellular location, and protein-protein interactions. In this review, we summarize the current understanding of the roles of SUMOylation and de-SUMOylation in DNA damage response (DDR) and DNA repair with a specific focus on the role of RPA SUMOylation in homologous recombination (HR).     

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.     

Chk1 has an essential role in the survival of differentiated cortical neurons in the absence of DNA damage

Neuronal death in the central nervous system contributes to the development of age-related neurodegeneration. The ATR/Chk1 pathway appears to function neuroprotectively to prevent DNA damage induced by cytotoxic agents. Here, we examine the function of Chk1 on cell viability of cortical neurons in the absence of additional DNA damaging stimuli. The Chk1-specific inhibitor, UCN-01, and the ATR inhibitor, Caffeine, cause neuronal apoptosis in differentiated neurons in the absence of additional treatment, whereas inhibition of ATM or Chk2, does not. UCN-01 treatment increased the detection of γ-H2AX phosphorylation, DNA strand breaks, and an activated p53-dependent DNA damage response (DDR), suggesting that Chk1 normally helps to maintain genomic stability. UCN-01 treatment also enhanced the apoptosis seen in neurons treated with DNA damaging agents, such as camptothecin (CPT). Our results indicate that Chk1 is essential for neuronal survival, and perturbation of this pathway increases a cell’s sensitivity to naturally accumulating DNA damage.     

Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders

The DNA damage response(DDR) is a complex biological system activated by different types of DNA damage.Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration,premature aging,and various types of cancers.Intriguingly,malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's,Alzheimer's,and Huntington's diseases.For many years,brain degenerative disorders were thought to result from aberrant neural death.Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells(astrocytes,microglia,and oligodendrocytes).Impairment in the functionality of glial cells results in pathological neuro-glial interactions that,in turn,generate a ‘‘hostile" environment that impairs the functionality of neuronal cells.These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.     

DNA damage response is suppressed by the high cyclin-dependent kinase 1 activity in mitotic mammalian cells

DNA damage response (DDR) is vital for genomic stability, and its deficiency is linked to tumorigenesis. Extensive studies in interphase (G(1)-S-G(2)) mammalian cells have revealed the mechanisms of DDR in great detail; however, how mitotic cells respond to DNA damage remains less defined. We report here that a full DDR is suppressed in mitotic mammalian cells until telophase/cytokinesis. Although early DDR markers such as the phosphorylations of ataxia telangiectasia mutated (ATM) and histone H2A.x (H2AX) can be readily detected, the ionizing radiation-induced foci (IRIF) formation of late DDR markers such as breast cancer type 1 susceptibility protein (BRCA1) and p53-binding protein 1 (53BP1) are absent until the telophase/cytokinesis stage. We further showed that the IR-induced ubiquitination cascade around DNA damage sites did not occur in mitotic cells, which explains, at least in part, why BRCA1 and 53BP1 cannot be recruited to the damaged sites. These observations indicate that DDR is suppressed in mitotic cells after the step of γH2AX formation. Not surprisingly, we found that the absence of a full DDR in mitotic cells was associated with the high cyclin-dependent kinase 1 (CDK1) activities. More 53BP1 IRIF could be detected when the irradiated mitotic cells were treated with a CDK1 inhibitor. Further, the activation of CDK5 in interphase cells impedes the formation of 53BP1 IRIF. Together, these results suggest that the DDR is suppressed by the high CDK1 activity in mitotic mammalian cells.     

Diverse functions of DNA glycosylases processing oxidative base lesions in brain

Endogenous and exogenous oxidative agents continuously damage genomic DNA, with the brain being particularly vulnerable. Thus, preserving genomic integrity is key for brain health and neuronal function. Accumulation of DNA damage is one of the causative factors of ageing and increases the risk of a wide range of neurological disorders. Base excision repair is the major pathway for removal of oxidized bases in the genome and initiated by DNA glycosylases. Emerging evidence suggest that DNA glycosylases have non-canonical functions important for genome regulation. Understanding canonical and non-canonical functions of DNA glycosylases processing oxidative base lesions modulating brain function will be crucial for the development of novel therapeutic strategies.     

Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.     

Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr^+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.     

The role of dePARylation in DNA damage repair and cancer suppression

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational modification regulating various biological pathways including DNA damage repair (DDR). Rapid turnover of PARylation is critically important for an optimal DNA damage response and maintaining genomic stability. Recent studies show that PARylation is tightly regulated by a group of enzymes that can erase the ADP-ribose (ADPR) groups from target proteins. The aim of this review is to present a comprehensive understanding of dePARylation enzymes, their substrates and roles in DDR. Special attention will be laid on the role of these proteins in the development of cancer and their feasibility in anticancer therapeutics.     

DNA double‐strand break response in stem cells: Mechanisms to maintain genomic integrity

Background

Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease.

Scope of Review

Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells.

Major Conclusions

ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin.

General Significance

The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Exome Capture Reveals ZNF423 and CEP164 Mutations, Linking Renal Ciliopathies to DNA Damage Response Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites?of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR. PAPERFLICK:     

The crosstalk between trace elements with DNA damage response,repair, and oxidative stress in cancer

DNA damage response (DDR) is a regulatory system responsible for maintaining genome integrity and stability, which can sense and transduce DNA damage signals. The severity of damage appears to determine DDRs, which can include damage repair, cell-cycle arrest, and apoptosis. Furthermore, defective components in DNA damage and repair machinery are an underlying cause for the development and progression of various types of cancers. Increasing evidence indicates that there is an association between trace elements and DDR/repair mechanisms. In fact, trace elements seem to affect mediators of DDR. Besides, it has been revealed that oxidative stress (OS) and trace elements are associated with cancer development. In this review, we discuss the role of some critical trace elements in the risk of cancer. In addition, we provide a brief introduction on DDR and OS in cancer. Finally, we will further review the interactions between some important trace elements including selenium, zinc, chromium, cadmium, and arsenic, and DDR, and OS in cancer.     

Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability

DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM's repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM's repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM's checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM's repair function is important for survival post irradiation.