Radiation-Induced DNA Breaks in Different Human Satellite DNA Sequence Areas, Analyzed by DNA Breakage Detection-Fluorescence<i>In Situ</i>Hybridization

Vázquez-Gundı́n, Fernando; Rivero, María Teresa; Gosálvez, Jaime; Fernández, José Luis Fernández

doi:10.1667/0033-7587(2002)157[0711:ridbid]2.0.co;2

Cited by 12 publications

(6 citation statements)

References 31 publications

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“…This possibility opens up a new question about heterogeneity for genome organization and mutation sensitivity for different genome domains. Experimental evidence shows that different genome domains may present different levels of susceptibility to DNA damage when exposed to equivalent external insults [14].…”

Section: Genome Organization and Protection In Reproductive Cellsmentioning

confidence: 99%

DNA Damage and Repair in Human Reproductive Cells

García-Rodríguez

Gosálvez

Agarwal

et al. 2018

IJMS

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The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of “stressors” that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.

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Section: Genome Organization and Protection In Reproductive Cellsmentioning

confidence: 99%

DNA Damage and Repair in Human Reproductive Cells

García-Rodríguez

Gosálvez

Agarwal

et al. 2018

IJMS

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“…The alkaline denaturation during the unwinding and electrophoretic steps produces ssDNA starting from the ends of the breaks, so more ssDNA is produced as the dose increases. At the macromolecular level, the local concentration of radical scavengers and different chromatin structures may influence the distribution of ionizing-radiation-induced damage [Vázquez-Gundín et al, 2002]. However, from a practical standpoint, damage can be considered to be randomly distributed when working with whole nucleus.…”

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confidence: 99%

Patterns of DNA migration in two‐dimensional single‐cell gel electrophoresis analyzed by DNA breakage detection‐fluorescence in situ hybridization

Rivero¹,

Vázquez-Gundı́n²,

Muriel³

et al. 2003

Environ and Mol Mutagen

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The single-cell gel electrophoresis (SCGE) or comet assay is a well-established procedure for quantifying DNA breakage and repair in single cells [Olive, 1999;Tice et al., 2000]. Nucleoids obtained after cell lysis of agarose-embedded cells are electrophoresed under alkaline or neutral conditions, and after staining DNA with a fluorescent dye, give the appearance of a comet with a head and a tail. DNA breaks increase the migration length and/or the amount of DNA in the tail. Not only DNA breaks, but also other types of lesions can be assessed, either as increases in migration, e.g., alkali-labile sites at alkaline pH, or as decreases in migration, e.g., DNA-DNA cross-links.Although widely used, there is no clear understanding of the electrophoretic behavior of the DNA in SCGE. In this communication we have performed neutral and alkaline electrophoresis sequentially on the same cell (i.e., a rightangled, bidimensional SCGE) to obtain information about the mechanisms of DNA migration. SCGE DNA patterns were evaluated using conventional fluorescence in situ hybridization (FISH) or DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) [Fernández and Gosálvez, 2002], since the hybridization signals result in a very sensitive delineation of DNA molecules.The study was carried out on human peripheral blood leukocytes, which were mixed with low-melting-point agarose to give an agarose concentration of 0.7%. The mix was pipetted onto a glass slide covered with a thin film of 0.65% standard agarose dried at 80°C and was covered with a glass coverslip. After the agarose solidified, the slide was exposed on ice to x-rays, by administering 0, 13, 26, and 39 Gy in transverse strips on the same slide. The slides were incubated sequentially for 30 min each in two lysing solutions, both at 22°C: 0.4 M Tris-HCl, 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), pH 7.5, followed by 0.4 M Tris-HCl, 2 M NaCl, 1% SDS, 0.05 M EDTA, pH 7.5. The first solution is sufficient for cell lysis; when both solutions are used separately, the first solution has a more pronounced effect than the second, even for spermatozoa. However, we have observed that using the two solutions sequentially increases the quality and sharpness of the chromatin and produces a better discrimination of the head of the comet. The slides then were washed extensively with TBE buffer (0.09 M Tris-borate, 0.002 M EDTA, pH 7.5), transferred to an electrophoresis chamber, and electrophoresed at 20 V (1 V/cm), 12 mA, 12.5 min, at 22°C in TBE buffer (first-run neutral electrophoresis of nondenatured DNA). After a wash in 0.9% NaCl, the slides were incubated at 22°C for 2.5 min in an alkaline unwinding solution (0.03 M NaOH, 1 M NaCl, pH 12.2) to generate singlestranded DNA (ssDNA) starting from the ends of DNA breaks. Next, the slides were washed extensively with 0.9% NaCl and were oriented 90°to the first electrophoresis for a

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“…To prevent as far as possible the possibility of ssDNA extending from DNA breaks generated near the ITRS sequence blocks, very restrictive unwinding conditions were employed. Under these conditions, the long ITRS blocks, measuring several hundred kilobases or megabases, are far longer than the ssDNA regions produced from the ends of the DNA breaks, practically ensuring that the quantification of the DNA damage is quite restricted to these specific targets (Vázquez‐Gundin et al, 2000, 2002). ITRSs were not found to be more damaged by X‐rays than was the genome overall, whereas damage from MNNG was 1.7 times greater.…”

Section: Discussionmentioning

confidence: 99%

“…In dose–response studies, the relative increase of the overall fluorescence intensity relative to the background signal from the same target in the untreated sample was plotted against dose. As established in previous work (Vázquez‐Gundín et al, 2002; Rivero et al, 2004), absolute values of intensity do not allow a comparison among different targets, due to the different target sizes and DNA probes, that is, different background signals. The use of the relative increase in signal overcomes this problem.…”

Section: Methodsmentioning

confidence: 95%

“…The terminal telomeric sequences from human cells are totally labeled by DBD‐FISH in control undamaged cells, and no increase in signal is detected despite X‐ray exposure or increased unwinding time. They probably behave as “breaks” from which the alkaline unwinding solution may initiate the production of ssDNA, being completely denatured because of their relatively small size, that is, 2.5–7.5 kb (Vázquez‐Gundín et al, 2002). Because the long ITRSs from Chinese hamster cells comprise hundreds of kilobases or megabases of DNA on each chromosome and because the length of ssDNA produced is much shorter than the target size, DNA damage should be detected confidently (Rivero et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to nitric oxide damage, and DNA‐PKcs has a specific local role in its repair

Mosquera¹,

Gosálvez²,

Sabatier³

et al. 2005

Genes Chromosomes & Cancer

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The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was used to analyze DNA single-strand breaks (SSBs) and alkali-labile sites induced by exposure to the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnomine hydrochloride (SIN-1) in the whole genome and in long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cells. The relative density of DNA damage generated in the ITRS by X-rays was similar to that induced in the genome overall, whereas it was 1.7 times higher when the alkylating agent MNNG was assayed. Nevertheless, after SNP or SIN-1 treatment, ITRSs proved to be 2.8 and 2.7 times relatively more damaged, respectively, than the whole genome. When the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was not active, as in XR-C1 mutant cells, the repair kinetics in the whole genome did not differ from that in the parental cell line with X-ray or SNP exposure. However, whereas the SSBs and alkali-labile sites induced in the ITRS by X-rays exhibited rejoining kinetics similar to that of the parental cell line, the damage induced by SNP was more slowly rejoined. This implies a role for DNA-PKcs in the repair of DNA damage induced by NO, especially in ITRSs. The results demonstrated intragenomic heterogeneity of NO-induced DNA damage and repair; there was a higher density of DNA damage in the ITRS blocks, possibly because of their guanine richness. This suggests that a parallel process may occur in the terminal telomeres, which has implications for premature aging and neoplastic development by chronic NO exposure in vivo.

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Radiation-Induced DNA Breaks in Different Human Satellite DNA Sequence Areas, Analyzed by DNA Breakage Detection-FluorescenceIn SituHybridization

Cited by 12 publications

References 31 publications

DNA Damage and Repair in Human Reproductive Cells

DNA Damage and Repair in Human Reproductive Cells

Patterns of DNA migration in two‐dimensional single‐cell gel electrophoresis analyzed by DNA breakage detection‐fluorescence in situ hybridization

Interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to nitric oxide damage, and DNA‐PKcs has a specific local role in its repair

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