Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of 2 weeks in vivo. We show that influenza induces DNA damage to both, when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67-positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.     

Biochemical aspects of radiation biology

In order to analyze the mechanisms of biological radiation effects, the events after radiation energy absorption in irradiated organisms have to be studied by physico-chemical and biochemical methods. The radiation effects in vitro on biomolecules, especially DNA, are described, as well as their alterations in irradiated cells. Whereas in vitro, in aqueous solution, predominantly OH radicals are effective and lead to damage in single moieties of the DNA, in vivo the direct absorption of radiation energy leads to 'locally multiply-damaged sites', which produce DNA double-strand breaks and locally denatured regions. DNA damage will be repaired in irradiated cells. Error free repair leads to the original nucleotide sequence in the genome by excision or by recombination. "Error prone repair"(mutagenic repair), leads to mutation. However, the biochemistry of these processes, regulated by a number of genes, is poorly understood. In addition, more complex reactions, such as gene amplification and transposition of mobile gene elements, are responsible for mutation or malignant transformation.     

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

The dual role model for p53 in maintaining genomic integrity

The tumour suppressor p53 is a potent mediator of cellular responses against genotoxic insults. In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53 actively participates in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery, and by its various biochemical activities. An important aspect in evaluating p53 functions is provided by the finding that the core domain of p53 harbours two mutually exclusive biochemical activities, sequence-specific DNA binding required for its transactivation function, and 3'-5' exonuclease activity, possibly involved in aspects of DNA repair. Based on the finding that modifications of p53 which lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its noninduced state, p53 should not be regarded as a 'dead' protein but, for example, via its exonuclease activity might be actively involved in prevention and repair of endogenous DNA damage. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. This dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.     

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.     

Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.     

DNA repair mechanisms in embryonic stem cells

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.     

DNA damage repair and transcription

Double-strand breaks arise frequently in the course of endogenous - normal and pathological - cellular DNA metabolism or can result from exogenous agents such as ionizing radiation. It is generally accepted that these lesions represent one of the most severe types of DNA damage with respect to preservation of genomic integrity. Therefore, cells have evolved complex mechanisms that include cell-cycle arrest, activation of various genes, including those associated with DNA repair, and in certain cases induction of the apoptotic pathway to respond to double-strand breaks. In this review we discuss recent progress in our understanding of cellular responses to DNA double-strand breaks. In addition to an analysis of the current paradigms of detection, signaling and repair, insights into the significance of chromatin remodeling in the double-strand break-response pathways are provided.     

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term “checkpoint” was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.     

ATM protein kinase: the linchpin of cellular defenses to stress

ATM is the most significant molecule involved in monitoring the genomic integrity of the cell. Any damage done to DNA relentlessly challenges the cellular machinery involved in recognition, processing and repair of these insults. ATM kinase is activated early to detect and signal lesions in DNA, arrest the cell cycle, establish DNA repair signaling and faithfully restore the damaged chromatin. ATM activation plays an important role as a barrier to tumorigenesis, metabolic syndrome and neurodegeneration. Therefore, studies of ATM-dependent DNA damage signaling pathways hold promise for treatment of a variety of debilitating diseases through the development of new therapeutics capable of modulating cellular responses to stress. In this review, we have tried to untangle the complex web of ATM signaling pathways with the purpose of pinpointing multiple roles of ATM underlying the complex phenotypes observed in AT patients.     

The role of repair in radiobiology

Apart from cancer and mutation induction, radiobiological effects on mammals are mostly attributable to cell 'death', defined as loss of proliferative capacity. Survival curves relate retention of that capacity to radiation dose, and often manifest a quasi-threshold ('shoulder'). The shoulder is attributable to an initial mechanism of repair ('Q-repair') which is gradually depleted as dose increases. Another form of repair, which is not depleted ('P-repair'), increases the dose required to deliver an average of one lethal event per cell (dose 'D0'). Neither form of repair can unambiguously be linked with repair of defects in isolated DNA. An important initial lesion may well be disruption of the complex structural relationship between the DNA, nuclear membrane and associated proteins. One form of P-repair may be restoration of that structural relationship.     

The nuclear γ-H2AX apoptotic ring: implications for cancers and autoimmune diseases

Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers and to the outcome of cancer chemotherapies, as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by “death receptors” and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, which microscopically looks like a nuclear annular staining early in apoptosis. This ring is, in three-dimensional space, a thick intranuclear shell consisting of epigenetic modifications including histone H2AX and DNA damage response (DDR) proteins. It excludes the DNA repair factors usually associated with γ-H2AX in the DDR nuclear foci. Here, we summarize our knowledge of the apoptotic ring, and discuss its biological and pathophysiological relevance, as well as its value as a potential pharmacodynamic biomarker for anticancer therapies.     

Alkyltransferase-like proteins: molecular switches between DNA repair pathways

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O ⁶-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here we review recent advances in unraveling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O ⁶-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway.     

DNA damage repair and transcription

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.