DNA double-strand breaks in mutagenesis

Several lines of research suggest that some systemic diseases, often associated with age-related conditions, may present with enhanced prevalences owing to very early influences on human development. This paper describes an analysis of 1264 adult Caucasian patients presenting either with primary open angle or narrow angle/angle closure glaucoma on the one hand, or with age-related cataract on the other. In addition, data on cataracts and primary open angle glaucoma on 254 patients of Caribbean origin and 190 of south east Asian origin were also examined. Patients were classified with respect to sex and season of birth. These variables can play a statistically significant role in the prevalence of glaucoma, which raises the possibility that environmental influences may be involved.     

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells

Recently the concept of dissociative identity disorder (formerly known as multiple personality disorder) has attracted increasing public and scientific interest. However, it is rarely diagnosed in the clinical setting. the reported case of a 47-year-old woman with a history of child abuse demonstrates the problems of differential diagnosis. A number of psychopathologic symptoms pointed to a multiple personality disorder, but in the follow-up psychotic symptoms such as delusions, possible hallucinations and bizarre behavior clearly emerged. The differential diagnosis of dissociative identity disorder includes paranoid schizophrenia, as in the case described, borderline personality disorder, hysteria, simulation and the false memory syndrome. Finally, social and cultural factors have to be considered.     

Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks

STATEMENT OF PROBLEM: The cone-screw abutment has been shown to diminish micromovement by reducing the burden of component loosening and fracture. However, anecdotal concern for cold welding of cone-screw joints in implant design has been identified as a potential source for lack of retrievability. PURPOSE: This comparative study evaluated the loosening torque, as a percentage of tightening torque, for the ITI Straumann and Astra Tech (3.5 and 4.0 mm diameters) implant systems, which use an 8-degree and 11-degree internal cone, respectively. MATERIAL AND METHODS: Implants and abutments from each system were mounted in a torque device, and a range of tightening torques was applied. Loosening torques were then measured, and the influence of conus angle, interfacial surface area, saliva contamination, and time delay to loosening were all assessed. RESULTS: The loosening torque only exceeded tightening torque at the highest levels, just before component failure, when plastic deformation was expected. For all clinically relevant levels of torque, both in a dry environment and with components bathed in artificial saliva at 37 degrees C, loosening torque was always seen to be 80% to 90% of tightening torque, demonstrating that cold welding does not occur. There was a high correlation between loosening and tightening torque for all systems tested, but no statistical difference when comparing wet versus dry or comparing individual data for each system. CONCLUSIONS: It can be concluded that for clinically relevant levels of tightening torque, no problems are anticipated with respect to retrievability.     

In vitro induction of primary DNA double-strand breaks in leukemic and normal blood cells by ultraviolet irradiation

The yield of UV-induced DNA double-strand breaks was studied for white blood cells ("light" fraction) derived from peripheral blood, and from patients with lymphomas, chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). The method employed was constant-field electrophoresis of plug-embedded DNA in agarose gel. Characteristic dose-response curves were obtained for various cell populations. Lymphoid cells, both from healthy subjects and CLL patients, revealed less damage to DNA under UV-irradiation, whereas CML cells were much more affected. Possible interpretation of these results includes species-specific differences in UV-induced DNA damage, as well as sufficient DNA crosslinking, thus interfering with DNA dsbs detection in irradiated cells.     

Non-random distribution of DNA double-strand breaks induced by particle irradiation

Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed.     

Rejoining of DNA single- and double-strand breaks in human white blood cells exposed to ionizing radiation

PURPOSE: To characterize inter- and intra-individual differences in X-ray-induced DNA strand break rejoining kinetics in human peripheral white blood cells (WBC) obtained from 10 healthy volunteers. MATERIALS AND METHODS: The alkaline and neutral versions of the comet assay were used to measure the rate of rejoining of predominantly single-strand breaks (ssb) following exposure to 8 Gy and double-strand breaks (dsb) following 75 Gy. RESULTS: All cells within a population responded in a similar fashion to induction of ssb and dsb; however, a subset of the WBC appeared to rejoin ssb more rapidly. For the 10 individuals examined, the percentage of ssb rejoined by the rapid component(s) was 47 +/- 16% and the rejoining half-time for the slow component was 1.3 +/- 0.4 h. By 24 h after 8 Gy, 4.9 +/- 3.8% of the initial ssb remained. For dsb rejoining, 58 +/- 11% of the initial damage was still present 4h after 75 Gy and by 24 h 32% of the initial level of damage was still detected. Heavily damaged cells present 24 h after 75 Gy varied from 4% to 50% and were excluded from the analysis of repair rates. CONCLUSIONS: Inter-individual variability exceeded intra-individual variability for 2 of 4 endpoints examined for ssb repair, but not for dsb repair. It was concluded that DNA damage measured using the comet assay could identify a range in the X-ray repair responses of WBC from different normal individuals. Whether these differences correlate with differences in cell killing by radiation remains to be determined.     

Hydroxyl radicals and DNA double-strand breaks in X-irradiated E. coli

We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.     

Induction and repair of double-strand breaks in the replicating DNA of HeLa cells

The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed.     

Joining of correct and incorrect DNA ends at double-strand breaks produced by high-linear energy transfer radiation in human fibroblasts

DNA double-strand breaks (DSBs) were measured within a 3.2-Mbp NotI fragment on chromosome 21 of cells of a normal human fibroblast cell line. Correct rejoining of DSBs was followed by measuring reconstitution of the original-size NotI fragment, and this was compared to total rejoining as measured by a conventional pulsed-field gel electrophoresis technique (FAR assay). After 80 Gy of particle irradiations with LETs in the range of 7-150 keV/microm, it was found that the repair kinetics was generally slower after irradiation with high-LET particles compared to X irradiation and that a larger proportion of the breaks remained unrepaired after 24 h. On the other hand, the misrejoining frequency as measured by the difference between correct and total rejoining after 24 h did not change with LET, but was approximately the same for all radiations at this dose, equal to 25-30% of the initial breaks. This result is discussed in relation to formation of chromosomal aberrations, deletion mutations and other biological end points.     

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: modeling the elution of DNA from nucleoids

A 36-year-old male with unspecific symptoms and normal physical examination had right cardiac enlargement on chest X-ray. Two-dimensional echocardiographic and thoracic computed tomography demonstrated an intracardiac mass. The tumor was surgically resected and the pathological diagnosis was mixed-type epicardial hemangioma. We discuss this case and review the literature.     

Apoptosis induced by X-rays and chemical agents in murine fibroblastic cell lines with a defect in repair of DNA double-strand breaks

This article describes the development and characteristics of a hospital-based lactation program and mother's own milk bank in a large pediatric hospital in the southwestern United States. Professional and technical staffing, physical space of the milk bank area, and the program's services and special features are outlined. Quality control issues about human milk preparation, fortification, storage, and transport are discussed.     

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage.     

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

DNA double-strand breaks, p53, and apoptosis during lymphomagenesis in scid/scid mice

The tumor-suppressing phenotype of p53 is thought to be due to its accumulation in response to DNA damage and resultant cell cycle arrest or apoptosis. scid/scid mice are defective in DNA double-strand break repair due to a mutation in DNA-dependent protein kinase (DNAPK). Treatment of scid/scid mice with gamma radiation or N-ethyl-N-nitrosourea resulted in approximately 86% incidence of T-cell lymphomas, compared with <6% in wild-type mice. The incidence of other tumor types was not increased in scid/scid mice, suggesting that the types of DNA double-strand break that are unrepaired in these mice are not strongly carcinogenic. To determine whether mutations in DNAPK and p53 interact, we examined mice deficient in both genes. Both scid/scid p53-/- and scid/scid p53+/- mice spontaneously developed lymphomas at shorter latency than did mice with either defect alone. Loss of the wild-type p53 allele was observed in 100% of tumors from scid/scid p53 +/- mice, indicating strong selection against p53. In contrast, p53 was not inactivated in lymphomas from scid/scid p53+/+ mice. Exposure of these tumor-bearing mice to gamma radiation resulted in p53 protein accumulation and high levels of apoptosis in all tumors that were not observed in tumors from scid/scid p53+/- mice. Thus, there was a bifurcation of molecular pathways to tumorigenesis. When p53 was heterozygous in the germ line, loss of the wild-type allele occurred, and the tumors became apoptosis resistant. When p53 was wild type in the germ line, p53 was not inactivated, and the tumors remained highly apoptosis sensitive.     

One-sided invasion events in homologous recombination at double-strand breaks

Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events.     

In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts

1. The identification of cytokine genes expressed in the central nervous system is critical to understanding the immune network in various diseases of brain, such as infection, degeneration, and malignancy. 2. Expression of cytokine genes in human astrocytoma cell lines and in fresh brain specimens was studied by the reverse-transcribed/polymerase chain reaction method. 3. The correlation between clinical malignancy and cytokine gene expression within malignant glioma was examined, especially regarding the relevancy of inhibitory cytokines, such as transforming growth factor-beta and interleukin-10.     

Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2(-) has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb+) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.     

Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.