The formation and repair of DNA breaks in subpopulations of irradiated rat bone marrow cells

Using the method of viscosimetry of alkaline lysates it was shown that the saturable DNA repair system was present in rat bone marrow cells which was described by a complex kinetics. Isokinetic fractionation in a sucrose and dextran gradient permitted to isolate three fractions differing in the ratio between differentiated cells and cells capable of division. The estimation of the postirradiation DNA repair kinetics in these cells revealed a comparatively high level of repair in normocytes which confirmed the presence of the relationship between the level of DNA repair in cells and their proliferative potency.     

Dynamic link of DNA demethylation,DNA strand breaks and repair in mouse zygotes

In mammalian zygotes, the 5‐methyl‐cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre‐replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as γH2A.X and PARP‐1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA‐methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.     

Inhibition of repair of DNA single strand breaks in mouse leukemia cells by actinomycin D

The effects of Actinomycin D, cytosine arabinoside and temperature shifts on the repair of single strand breaks produced in murine leukemia cell DNA by ionizing radiation have been studied. A recently introduced modification of the alkaline sucrose sedimentation methods was used, allowing breaks to be demonstrated following clinical range irradiation doses. The results contrast to previous data using standard gradient procedures and indicate that low concentrations of Actinomycin D can inhibit single strand break repair, while cytosine arabinoside is ineffective. Inhibition can also be demonstrated by temperature shifts to 3° but not 24°, paralleling previous results from cellular repair studies (Elkind-Sutton repair). The results are consistent with the hypothesis that the accumulation of sublethal radiation damage in mammalian cells may be based on residual non-repaired single strand breaks.     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.     

Chromatin dynamics and the repair of DNA double strand breaks

DNA double-strand breaks (DSBs) arise through both replication errors and from exogenous events such as exposure to ionizing radiation. DSBs are potentially lethal, and cells have evolved a highly conserved mechanism to detect and repair these lesions. This mechanism involves phosphorylation of histone H2AX (γH2AX) and the loading of DNA repair proteins onto the chromatin adjacent to the DSB. It is now clear that the chromatin architecture in the region surrounding the DSB has a critical impact on the ability of cells to mount an effective DNA damage response. DSBs promote the formation of open, relaxed chromatin domains which are spatially confined to the area surrounding the break. These relaxed chromatin structures are created through the coupled action of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. The resulting destabilization of nucleosomes at the DSB by Tip60 and p400 is required for ubiquitination of the chromatin by the RNF8 ubiquitin ligase, and for the subsequent recruitment of the brca1 complex. Chromatin dynamics at DSBs can therefore exert a powerful influence on the process of DSB repair. Further, there is emerging evidence that the different chromatin structures in the cell, such as heterochromatin and euchromatin, utilize distinct remodeling complexes and pathways to facilitate DSB. The processing and repair of DSB is therefore critically influenced by the nuclear architecture in which the lesion arises.Key words: p400, chromatin remodeling, DNA repair, NuA4, H2AX, acetylation, nucleosome, tip60Damage to cellular DNA can occur through multiple pathways, including exposure to genotoxic agents, the production of endogenous reactive oxygen species or errors which arise during DNA replication. To combat this continuous assault on the genome, mammalian cells have evolved multiple DNA repair pathways. The most challenging lesions to repair are DSBs, which physically cleave the DNA strand. DSBs can occur through exposure to IR, the collapse of replication forks or during the processing of certain types of DNA damage. Over the last 20 years, a clear picture of how the cell detects and repairs DSBs has emerged.^1,2 The earliest event in the cell''s response to DSBs is the rapid recruitment of the ATM kinase, followed by the phosphorylation of histone H2AX (termed γH2AX) on large chromatin domains which extend for 100''s of kilobases on either side of the DSB.³ The mdc1 scaffold protein is then recruited to γH2AX,⁴ providing a docking platform for the recruitment and retention of additional DNA repair proteins, including the MRN complex, the RNF8 ubiquitin ligase and the brca1 and 53BP1 proteins, onto the chromatin at DSBs.^5–7 Eventually, this spreading of DNA repair proteins along the chromatin from the DSB leads to the formation of IRIF, which can be visualized by immunofluorescent techniques. DSBs are then repaired by NHEJ, in which broken DNA ends are directly religated, or by HR, using the undamaged sister chromatid (present during S-phase) as a template. A defining characteristic of DSB repair is the dominant role that chromatin structure plays in the detection and repair of these lesions. In this review, we will examine recent work exploring how remodeling of the chromatin structure adjacent to DSBs plays a key role in the repair of DSBs.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

The cellular response to general and programmed DNA double strand breaks

DNA double strand breaks (DSBs) are among the most dangerous lesions that can occur in the genome of eukaryotic cells. Proper repair of chromosomal DSBs is critical for maintaining cellular viability and genomic integrity and, in multi-cellular organisms, for suppression of tumorigenesis. Thus, eukaryotic cells have evolved specialized and redundant molecular mechanisms to sense, respond to, and repair DSBs. In this chapter, we provide an overview of the progress that has been made over the last decade in elucidating the identity and function of components that participate in the cellular response to chromosomal DSBs. Then, we discuss, in more depth, the response to DSBs that occur in the context of the V(D)J recombination and IgH class switch recombination reactions that occur in cells of the lymphocyte lineage.     

DNA strand breaks in pancreatic islets by in vivo administration of alloxan or streptozotocin

Administration of diabetogenic doses of alloxan or streptozotocin to rats caused extensive DNA strand breaks in pancreatic islets. DNA of pancreatic exocrine cells was not affected by either alloxan or streptozotocin. hepatocyte DNA was fragmented by streptozotocin but not by alloxan. Intracellular NAD level was decreased in tissues whose DNA was fragmented. The results may raise a novel aspect concerning the mechanisms of action of the diabetogenic agents as well as concerning the organotropisms of the agents.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a “constant ^·OH radical scavenging environment”, k of 1.5 × 10⁷ s^-1 by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0 ± 0.2 at [F] < 100 μM by a mechanism other than through direct scavenging of ^·OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H^· atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from ^·OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.     

Assessment of radiation-induced DNA strand breaks and their repair in unlabeled cells.

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.     

Evaluation of DNA double strand breaks repair efficiency in head and neck cancer

Head and neck cancers (head and neck squamous cell carcinomas [HNSCC]) are a heterogeneous group of neoplasms with varying presenting symptoms, treatment, and expected outcome. There is a need to find an effective way of its treatment at the molecular level. Thus, we should identify the mechanism of cancer cell response to damaging agents' activity, especially at DNA level. Our major goal was to evaluate the efficacy of DNA double strand breaks (DSBs) repair in HTB-43 and SCC-25 cancer cell lines as well as lymphocytes taken from HNSCC patients and healthy donors. The DNA repair efficiency was measured by neutral comet assay as well as extrachromosomal assay for DNA DSBs repair (TAK assay). We determined the levels of two main pathways of DNA DSBs-nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). Neutral comet assay was used for evaluation of DNA DSBs repair after treatment with genotoxic agents. DNA DSBs induced by gamma radiation were repaired slower in lymphocytes from HNSCC patients than in lymphocytes from healthy controls. HTB-43 and SCC-25 cancer cell lines have higher efficacy of NHEJ and HRR than lymphocytes taken from patients as well as control subjects. Our results confirm the necessity of further studies on the mechanisms of DNA DSBs repair to provide insight into the molecular basis of head and neck cancer, which will allow us to improve methods of HNSCC treatment.     

Detection in situ of gamma-ray-induced DNA strand breaks in single cells: enzymatic labelling of free 3'-OH ends

We report a procedure allowing the detection and counting of free 3'-OH DNA strand extremities in single cells in situ. Terminal transferase (TdT) catalysed the incorporation of 3H-dGMP into fixed nuclei of human colonic adenocarcinoma cells (HT29), using free 3'-OH ends as initiator. Radioactivity was detected by autoradiography and determined quantitatively with a rapid image-processing system for grain counting. The initiator activity for TdT increases with the dose of gamma-rays in the dose range 2.5-20 Gy.