Preserving genome integrity and function: the DNA damage response and histone modifications

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.     

Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage

The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity

As obligate phototrophs, plants harness energy from sunlight to split water, producing oxygen and reducing power. This lifestyle exposes plants to particularly high levels of genotoxic stress that threatens genomic integrity, leading to mutation, developmental arrest and cell death. Plants, which with algae are the only photosynthetic eukaryotes, have evolved very effective pathways for DNA damage signalling and repair, and this review summarises our current understanding of these processes in the responses of plants to genotoxic stress. We also identify how the use of new and emerging technologies can complement established physiological and ecological studies to progress the application of this knowledge in biotechnology.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

DNA repair in ultraviolet-irradiated HeLa cells is disrupted by aphidicolin: The inhibition of repair need not imply the absence of repair synthesis

Aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase α, has been reported to inhibit repair DNA synthesis in ultraviolet-irradiated, normal human fibroblasts but not in HeLa cells. By the use of assays for repair other than the measurement of repair synthesis, it is shown here that repair in HeLa cells is in fact susceptible to aphidicolin. Severe inhibition of DNA repair, with failure of individual repair events to be completed, and a smaller number of lesions removed, can occur even though repair synthesis continues.     

Inhibition of poly(ADP-ribosyl)ation in cancer: Old and new paradigms revisited

Inhibitors of poly(ADP-ribose) polymerases actualized the biological concept of synthetic lethality in the clinical practice, yielding a paradigmatic example of translational medicine. The profound sensitivity of tumors with germline BRCA mutations to PARP1/2 blockade owes to inherent defects of the BRCA-dependent homologous recombination machinery, which are unleashed by interruption of PARP DNA repair activity and lead to DNA damage overload and cell death. Conversely, aspirant BRCA-like tumors harboring somatic DNA repair dysfunctions (a vast entity of genetic and epigenetic defects known as “BRCAness”) not always align with the familial counterpart and appear not to be equally sensitive to PARP inhibition. The acquisition of secondary resistance in initially responsive patients and the lack of standardized biomarkers to identify “BRCAness” pose serious threats to the clinical advance of PARP inhibitors; a feeling is also emerging that a BRCA-centered perspective might have missed the influence of additional, not negligible and DNA repair-independent PARP contributions onto therapy outcome. While regulatory approval for PARP1/2 inhibitors is still pending, novel therapeutic opportunities are sprouting from different branches of the PARP family, although they remain immature for clinical extrapolation. This review is an endeavor to provide a comprehensive appraisal of the multifaceted biology of PARPs and their evolving impact on cancer therapeutics.     

Structural and functional characterization of the MERIT40 to understand its role in DNA repair

MERIT40 (MEdiator of RAP80 Interaction and Targeting 40) is a novel associate of the BRCA1-complex and plays an essential role in DNA damage repair. It is the least characterized protein of BRCA1-complex and mainly responsible for maintaining the complex integrity. However, its structural and functional aspects of regulating the complex stability still remain elusive. Here, we carried out a comprehensive examination of MERIT40 biophysical properties and identified its novel interacting partner which would help to understand its role in BRCA1-complex. The recombinant protein was purified by affinity chromatography and unfolding pathway was determined using spectroscopic and calorimetric methods. Molecular model was generated using combinatorial approaches of modeling, and monomer–monomer docking was carried out to identify dimeric interface. Disordered region of MERIT40 was hatchet using trypsin and chymotrypsin to illustrate the existence of stable domain whose function was speculated through DALI search. Our findings suggest that MERIT40 forms a dimer in a concentration-independent manner. Its central region shows remarkable stability towards the protease digestion and has structural similarity with vWA-like region, a domain mainly present in complement activation factors. MERIT40 undergoes a three-state unfolding transition pathway with a dimeric intermediate. It interacts with adaptor molecule of BRCA1-complex, called ABRAXAS, thus help in extending the bridging interaction among various members which further stabilizes the whole complex. The results presented in this paper provide first-hand information on structural and folding behavior of MERIT40. These findings will help in elucidating the role of protein–protein interactions in stabilization of BRCA1-complex.     

Poly(ADP-ribose)polymerase-1 is required for integration of the human immunodeficiency virus type 1 genome near centromeric alphoid DNA in human and murine cells

This study examined the efficiency of human immunodeficiency virus type 1 (HIV-1) integration in poly(ADP-ribose)polymerase-1 (PARP-1)-deficient murine cells and in human cell lines transfected with small interfering RNA against PARP-1 (PARP-1 siRNA). To semi-quantify the amount of integrated HIV-1 genome, real-time nested PCR was carried out using primers specific for Alu and alphoid DNA combined with primers for the HIV-1 genome. The results showed that the integration efficiency of the HIV-1 genome near Alu DNA, which is randomly distributed in the chromosome, is reduced in PARP-1-deficient murine cells, but not in PARP-1 siRNA-transfected human cells. By contrast, the integration efficiency of the HIV-1 genome near alphoid DNA, which is localized in the centromere region, is significantly reduced in PARP-1-deficient murine cells and in PARP-1 siRNA-transfected human cells. These results suggest that PARP-1 is required for HIV-1 integration near the centromere region both in human and murine cells.     

Molecular interaction analysis of cigarette smoke carcinogens NNK and NNAL with enzymes involved in DNA repair pathways: An in silico approach

DNA damage occurs almost all the times in cells, but is repaired also continuously. Occurrence of all these mutations and their accumulation in one cell which finally becomes tumorigenic/carcinogenic appears possible if the DNA repair mechanism is hampered. We hypothesize that alterations in DNA repair pathways, either all or at least at one i.e. genetic, translational or posttranslational level, becomes quite imperative for the initiation and progression of Cancer. Therefore, we investigated the interaction capability of some carcinogens with the enzymes involved in the DNA repair mechanisms. Cigarette smoke''s derivatives like NNK and NNAL are well established carcinogens. Hence, we analyzed 72 enzymes involved in the DNA repair Mechanisms for their interactions with ligands (NNK and NNAL). The binding efficiencies with enzymes ranging from +36.96 to -7.47 Kcal/Mol. Crystal Structure of Human Carbonmonoxy-Haemoglobin at 1.25 Å Resolution, PDB ID-1IRD as a +Ve control, showed binding energy -6.31 to -6.68 Kcal/Mol. and Human heat shock factor-binding protein 1, PDB ID- 3CI9 as a -Ve control, showed - 3.91 to +2.09 Kcal/Mol. Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. Study indicated the loss of functions of these enzymes, which probably could be a reason for fettering of DNA repair pathways resulting in damage accumulation and finally cancer formation.     

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: Comparison with quercetin

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells’ capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.     

Function of poly(ADP-ribose) polymerase in response to DNA damage: Gene-disruption study in mice

To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, [EC 2.4.2.30]) in DNA damage responses, genetic and biochemical approaches were undertaken. By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality. PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to -irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage. p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA. However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following -irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade. On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA. Human PARP expressed in E. coli showed a similar effect on pRB-phosphorylation activity of cdk2. These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest.     

Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2''deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.     

The function of poly (ADP-ribosylation) in DNA breakage and rejoining

Poly (ADP-ribose) polymerase has an obligatory requirement for DNA strand-breaks in order to show full enzyme activity. Exposure of cells to DNA damaging agents activates this enzyme presumably through the production of DNA strand-breaks, either directly or via cellular enzymes. Recent evidence from manipulations of the cloned cDNA of this enzyme confirm the earlier evidence, obtained using enzyme inhibitors, that this enzyme is involved in DNA excision repair, probably at or near the ligation step. A very unusual human genetic disease has provided direct evidence for a link between the enzyme activities of poly (ADP-ribose) polymerase and of DNA ligase I. There is also some evidence that this enzyme may be involved in other cases of DNA breakage and rejoining, such as homologous and non-homologous DNA recombination, for example, in sister chromatid exchanges, in DNA transfection, in the intergration of retroviral proviral DNA and in variable antigen switching in African trypanosomes.     

Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.