Radiation sensitivity of lymphocytes from healthy individuals and cancer patients as measured by the comet assay

Lymphocytes of healthy volunteers (n=24) and of tumour patients (n=30, 18 of whom had experienced severe side-effects) were irradiated with x-rays in vitro. DNA damage was analysed after 0.25–2 Gy and DNA repair after 2 Gy, and quantification of both endpoints was done by the comet assay. The individual differences in radiation-induced DNA damage as well as in the repair kinetics were observed to be striking for both healthy donors and tumour patients. After a repair time of 3 h, following 2 Gy x-irradiation, some of the healthy volunteers showed no residual DNA damage at all in their lymphocytes, whereas others revealed about 30%. There was no indication that our results were affected by either age, gender or smoking habits. Slow repair kinetics and high amounts of residual damage were characteristic for many but not all tumour patients who had experienced severe side-effects in their normal tissues during or after radiotherapy (n=18). Our conclusion is that those individuals showing poor DNA repair characteristics in the lymphocytes following in vitro irradiation, have a high probability of being radiosensitive. The opposite conclusion is not necessarily true: if repair is effective, this does not mean that the individual is radioresistant, because factors other than impaired repair may cause radiosensitivity. Received: 3 May 2000 / Accepted: 1 December 2000     

Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells

Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.     

XPD Gene rs13181 Polymorphism and DNA Damage in Human Lymphocytes

Genetic heterogeneity influencing enzyme activity may change the capacity to repair DNA damage induced by environmental and endogenous factors. This study aims to assess the impact of Lys751Gln (A/C) polymorphism in the XPD gene, encoding an enzyme involved in the nucleotide excision repair pathway, on individual DNA damage. The DNA damage in human lymphocytes (% DNA in the tail) was quantified by means of single-cell gel electrophoresis. Baseline levels of DNA damage significantly differ between AA homozygotes and carriers of the C allele, and the observed differences were not related to age, gender, or smoking status. It seems that the AA variant is associated with enhanced protection against oxidative DNA damage.     

Replication protein A phosphorylation and the cellular response to DNA damage

Defects in cellular DNA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and basal DNA repair have been implicated as causal factors in diseases with DNA instability like cancer, Fragile X and Huntington's. Replication protein A (RPA) is essential for multiple processes in DNA metabolism including DNA replication, recombination and DNA repair pathways (including nucleotide excision, base excision and double-strand break repair). RPA is a single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA binds ssDNA with high affinity and interacts specifically with multiple proteins. Cellular DNA damage causes the N-terminus of the 32-kDa subunit of human RPA to become hyper-phosphorylated. Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes. This suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.     

Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage

Neuronal death can be induced by DNA-damaging agents and occurs by apoptosis involving a specific signal-transduction pathway. However, to our knowledge, methods for the quantitative determination of DNA damage in individual neurons have not yet been described. Here we optimize the single-cell gel electrophoresis (SCGE) or "comet"-assay to measure DNA damage within individual neurons growing in dissociated cell culture. In addition, we have written a macro for the NIH Image program to determine the tail moment of individual comets. We have calibrated this method using gamma-irradiated (0-16 Gy) cerebral cortical neurons from the rat central nervous system. Neuronal DNA damage (in the form of DNA strand breaks) occurs in a linear, dose-dependent manner, which can be quantitatively determined in vitro using the SCGE assay. These data demonstrate that the SCGE assay is an effective method to measure DNA damage in individual neurons and may be highly useful for the study of neuronal DNA damage formation, repair and apoptosis.     

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organisation of repair

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the "random collision" model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a "processive" mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. These two models give different predictions concerning the time course of repair. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate of repair until scanning is complete. We have examined the time course of DNA repair in human fibroblasts given low (generally sublethal) doses of UV light. Using 3 distinct assays, we find no sign of a constant repair rate after 4 J/m2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. The implications of this finding for the structural organisation of repair are discussed.     

Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity

The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity.Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways.Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p = 0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by β-cryptoxanthin and β-tocopherol (p < 0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p = 0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p < 0.05).We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity.     

Crosstalk between the nucleotide excision repair and Fanconi anemia/BRCA pathways

Cells have evolved multiple distinct DNA repair pathways to efficiently correct a variety of genotoxic lesions, and decades of study have led to an improved understanding of the mechanisms and regulation of these individual pathways. However, there is now an increasing appreciation that extensive crosstalk exists among DNA repair pathways and that this crosstalk serves to increase the efficiency and diversity of response to damage. The Fanconi anemia (FA)/BRCA and nucleotide excision repair (NER) pathways have been shown to share common factors, and often work in concert to repair damage. Genomic studies are now revealing that many tumors harbor somatic mutations in FA/BRCA or NER genes, which may provide a growth advantage, but which could also be exploited therapeutically.     

Length-dependent processing of telomeres in the absence of telomerase

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.     

Evidence that DNA polymerases alpha and beta participate differentially in DNA repair synthesis induced by different agents

The roles of DNA polymerases alpha and beta in DNA replication and repair synthesis were studied in permeable animal cells, using different agents to induce repair synthesis. DNA polymerase inhibitors were used to investigate which polymerases were involved in repair synthesis and in replication. Polymerase alpha was responsible for replication. On the other hand, both polymerases alpha and beta were involved in DNA repair synthesis; the extent to which each polymerase participated depended primarily on the agent used to damage DNA. Polymerase beta was primarily responsible for repair synthesis induced by bleomycin or neocarzinostatin, whereas polymerase alpha played a more prominent role in repair synthesis indiced by N-methyl-N'-nitro-N-nitrosoguanidine or N-nitrosomethyl urea. More DNA damage was induced by the alkylating agents than by bleomycin or neocarzinostatin, suggesting that the extent of involvement of polymerase alpha or beta in DNA repair synthesis is related to the amount or type of DNA damage. In addition, salt concentration was found to have little or no effect on the results obtained with the DNA polymerase inhibitors. Our findings provide an explanation for conflicting reports in the literature concerning the roles of DNA polymerases alpha and beta in DNA repair.     

DNA repair: relationship to drug and radiation resistance, metastasis and growth factors

DNA repair mechanisms are important for the recovery of both normal and malignant tissues from radiation and chemotherapy. Drug 'resistance' may merely reflect the similarity of cancer to normal tissues. Investigating the normal repair mechanisms by cloning human DNA repair genes will permit a much better comparison. Therapeutic inhibition of DNA repair may be possible with poly-ADP-ribose polymerase inhibitors. A differential effect may be obtained since less-differentiated cells have a higher poly-ADP-ribose polymerase activity. Clinical application of repair inhibitors can be achieved by using antimetabolites such as high-dose hydroxyurea which produces levels of 1-3 mmol litre -1/24 hours. The whole cell and tissue response to DNA damage is more complex than removal of adducts and joining strand breaks. DNA damage can result in an increase in growth-factor receptors, the release of soluble mediators that affect undamaged cells and stimulation of plasminogen activator. These changes may enhance growth and recovery as well as bypass or repair the damage. The generation of heterogeneity in a tumour population may be mediated by DNA rearrangements. Genetic instability is much higher in metastatic clones and a comparison of DNA strand-break repair in a metastatic and a non-metastatic line showed more rapid repair in the former. Aberrant use of DNA repair stimulated by growth factors may mediate tumour progression and heterogeneity as well as drug resistance.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.