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PubMed2025

Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.

A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.

DNA Breaks, Double-Stranded↗

PubMed2017

And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break.

Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Animals↗

PubMed2005

Roles of double-strand breaks, nicks, and gaps in stimulating deletions of CTG.CAG repeats by intramolecular DNA repair.

A series of plasmids harboring CTG.CAG repeats with double-strand breaks (DSB), single-strand nicks, or single-strand gaps (15 or 30 nucleotides) within the repeat regions were used to determine their capacity to induce genetic instabilities. These plasmids were introduced into Escherichia coli in the presence of a second plasmid containing a sequence that could support homologous recombination repair between the two plasmids. The transfer of a point mutation from the second to the first plasmid was used to monitor homologous recombination (gene conversion). Only DSBs increased the overall genetic instability. This instability took place by intramolecular repair, which was not dependent on RuvA. Double-strand break-induced instabilities were partially stabilized by a mutation in recF. Gaps of 30 nt formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nt did not induce expansions or deletions. Formation of this deletion product required the CTG.CAG repeats to be present in the single-stranded region and was stimulated by E.coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the intramolecular repair-induced instabilities and the formation of the 30 nt deletion product.

Base Sequence↗

PubMed2006

Clusters of DNA double-strand breaks induced by different doses of nitrogen ions for various LETs: experimental measurements and theoretical analyses.

The yields and clustering of DNA double-strand breaks (DSBs) were investigated in normal human skin fibroblasts exposed to gamma rays or to a wide range of doses of nitrogen ions with various linear energy transfers (LETs). Data obtained by pulsed-field gel electrophoresis on the dose and LET dependence of DNA fragmentation were analyzed with the randomly located clusters (RLC) formalism. The formalism considers stochastic clustering of DSBs along a chromosome due to chromatin structure, particle track structure, and multitrack action. The relative biological effectiveness (RBE) for the total DSB yield did not depend strongly on LET, but particles with higher LET produced higher fractions of small DNA fragments, corresponding in the formalism to an increase in the average number of DSBs per DSB cluster. The results are consistent with the idea that DSB clustering along chromosomes is what leads to large RBEs of high-LET radiations for major biological end points. At a given dose, large fragments are less affected by the variability in LET than small fragments, suggesting that the two free ends in large fragments are often produced by two different tracks. The formalism successfully described an extra increase in small DNA fragments as dose increases and a related decrease in large fragments, mainly due to interlacing of DSB clusters produced along a chromosome by different tracks, since interlacing cuts larger DNA fragments into smaller ones.

Cell Line↗

PubMed2000

The effects of microgravity on ligase activity in the repair of DNA double-strand breaks.

PURPOSE: In recent years, contradictory data have been reported about the effects of microgravity on radiation-induced biological responses in space experiments. The aim of the present study was to clarify whether enzymatic repair of DNA double-strand breaks is affected by microgravity using an in vitro enzymatic reaction system. MATERIALS AND METHODS: The DNA repair activity of T4 DNA ligase (EC 6.5.1.1) was measured in vitro for a DNA substrate damaged by restriction enzyme digestion during a US Space Shuttle mission (Discovery; STS-91). After the flight, the amount of ligated DNA molecules was measured using an electrophoresis method. RESULTS: Ligated products (closed circular DNA, open circular DNA and multimeric ligated products) were produced by T4 DNA ligase treatment of linear DNA containing double-strand breaks, and they increased with increasing T4 DNA ligase concentration (0-3 units per microg of plasmid DNA). Almost no difference in T4 DNA ligase activity was detected between the space experiments and the control ground experiments. CONCLUSIONS: No significant effect of microgravity on ligation of damaged DNA was found during space flight. Therefore, other mechanisms must account for the synergism between radiation and microgravity, if it exists.

DNA Damage↗

PubMed2005

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients.

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.

Antigens, Nuclear↗

PubMed2025

Efficiency of DNA repair mechanisms of domestic dog primary fibroblasts isolated from small and large breeds of different ages in response to double stranded breaks (DSB).

Aging is associated with increased genomic instability, a phenomenon largely driven by the accumulation of DNA damage over time, and large species of mammals seem to have more robust DNA repair systems associated with longer lives. Among DNA lesions, double-strand breaks (DSBs) are particularly deleterious and have been implicated in age-related functional decline and disease. In this study, we investigated how age and body mass affect the efficiency of DSB repair (DSBr) in primary fibroblast cells isolated from domestic dogs, a species that exhibits significant intraspecies variation in lifespan and body mass. Primary fibroblast cells were isolated from puppies and senior dogs of both large and small breeds. Cells were treated with 100 µM etoposide to induce DSBs and subsequently analyzed at two post-treatment recovery intervals (2 and 24 h) to correlated with the two pathways associated with DSBr, the fast, non-homologous end joining (NHEJ) and the much slower, homologous recombination (HR). Cells were stained for γ-H2AX foci and images were collected using confocal microscopy. We found that mean fluorescence per cell was higher in older dogs of both size classes in the 2 h recovery, indicating higher amounts of DNA damage but suggesting similar efficiencies through the NHEJ repair mechanism in older dogs despite size class. We also show that mean fluorescence per cell was higher in the older large breed dogs in the 24 h recovery, suggesting that the slower phase associated with HR seems to be deficient in cells from older, larger breeds of dogs. These findings support the broader theory that aging is associated with impaired genomic maintenance and establish domestic dogs as a valuable model for studying the cellular mechanisms of age-related genomic instability.

Animals↗

PubMed2000

Formation and repair of DNA double-strand breaks in gamma-irradiated K562 cells undergoing erythroid differentiation.

Cellular differentiation is accompanied by gross changes in nuclear organization, metabolic pathways and gene expression characteristics. To investigate, whether the response to radiation damage is altered during cellular differentiation, we studied the formation and repair of DNA double-strand breaks in gamma-irradiated K562 erythroleukemia cells induced to differentiate by exposure to butyric acid. We applied an assay based on pulsed-field gel electrophoresis and Southern hybridization to measure break induction in several genomic restriction fragments. Pulsed-field gel electrophoresis of (14)C-labelled unrestricted DNA was used to study the rejoining of gamma-radiation-induced breaks in the whole genome. Total rejoining and joining of correct break ends in specific genomic regions was monitored by hybridization analysis of blots of unrestricted and restriction digested DNA with single-copy probes. The yields of gamma-ray-induced DNA double-strand breaks were found to decrease with differentiation by about 20%. Correct rejoining of radiation-induced breaks, as measured by the reconstitution of broken restriction fragments, was unaltered in differentiating cells compared to actively proliferating precursor cells. Total rejoining, however, appeared to be retarded in differentiating cells. The results suggest that in spite of the fundamental changes accompanying differentiation, the cellular damage response pathways are not essentially affected throughout erythroid differentiation.

Butyrates↗

PubMed1996

Capture of retrotransposon DNA at the sites of chromosomal double-strand breaks.

Non-homologous repair of broken chromosomes in Saccharomyces cerevisiae can be studied at a defined location by expressing the site-specific HO endonuclease that cuts the mating-type (MAT) locus. When homologous recombination is prevented, most double-strand breaks are repaired by non-homologous end-joinings similar to those observed in mammalian cells. About 1% of non-homologous repair events were exceptional, having 'captured' approximately 100 base pairs of DNA within the HO cleavage site. In each case, the insertion came from yeast's retrotransposon Tyl element. Four of the five contained the R-U5 region, which is the first part of Tyl messenger RNA to be converted to complementary DNA. The capture of cDNA fragments at the sites of double-strand breaks may account for the way that pseudogenes and long and short interspersed sequences (LINES and SINES) have been inserted at many locations in the mammalian genome.

Base Sequence↗

PubMed1998

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.

Animals↗

PubMed2001

DNA double-strand breaks in mouse kidney cells with age.

A Biojector device fitted with a CO2 cartridge was used to prepare single cell suspensions from kidneys of 12-month- (middle-aged) and 24-month-old (old) C57B1/6 mice. Microgel electrophoresis of DNA from these cells revealed a modest but significant 7.3% increase (P = 0.04) in DNA double-strand breaks in old mice. This increase is equivalent to the DNA damage induced by 0.1 Gray of X-rays (5 double-strand breaks) in kidney cells of 10-month-old mice, as determined by a standard calibration curve. Greater DNA damage with aging was also positively correlated with higher levels of pathology in the kidneys.

Aging↗

PubMed1989

Double-strand breaks at an initiation site for meiotic gene conversion.

It has been proposed that the initiation of meiotic recombination involves either single-strand or double-strand breaks in DNA. It is difficult to distinguish between these on the basis of genetic evidence because they give rise to similar predictions. All models invoke initiation at specific sites to explain polarity, which is a gradient in gene conversion frequency from one end of a gene to the other. In the accompanying paper we describe the localization of an initiation site for gene conversion to the promoter region of the ARG4 gene of the yeast Saccharomyces cerevisiae. Here, we show that a double-strand break appears at the ARG4 recombination initiation site at the time of recombination, and that the broken DNA molecules end in long single-stranded tails.

Gene Conversion↗

PubMed1994

Enhanced chromosomal response of ataxia-telangiectasia cells to specific types of DNA double-strand breaks.

The chromosomal response of two ataxia-telangiectasia (A-T) lymphoblastoid cell lines (A-T-PA and A-T-KM) to restriction endonucleases (RE) is compared with that of a normal (N-SW) lymphoblastoid cell line. The RE used were PvuII (generating DNA double-strand breaks with blunt termini), BamHI (cohesive termini with 4 base, 5' overhangs) and PstI (cohesive termini with 4 base 3' overhangs). Chromatid aberrations were analysed in cells 5 h after treatment. Cells were porated using streptolysin O to allow entry of RE. Both A-T lines showed an enhanced frequency of chromatid breaks in G2 phase compared with normal cells in response to RE. The enhanced response of A-T cells was most marked in the case of PvuII treatment when the enhancement ratios were 2.5 and 4.2 for A-T-PA and A-T-KM respectively. However, the frequency of DNA double-strand breaks (dsb), measured by neutral filter elution, were considerably lower in A-T-PA cells than N-SW, due to a lower efficiency of poration. When A-T-PA cells were treated with streptolysin O at a higher concentration (0.3 Units/ml), a condition that apparently led to a similar level of poration in A-T-PA as in N-SW cells treated with 0.06 Units/ml as judged by the similar number of dsb induced in the two lines for a given PvuII concentration, the enhancement ratio for A-T-PA cells treated with PvuII increased from 2.5 to 5.8. BamHI and PstI were found to be less clastogenic in all three cell lines as found previously for Chinese hamster cells, although part of this effect may be due to a lower activity, particularly in the case of PstI. However, even at a 4-6-fold higher concentration, BamHI was still less clastogenic than PvuII. It is concluded that dsb with blunt termini are more clastogenic than those with cohesive termini. The results suggest that the chromosomal sensitivity of A-T cells may result from a defect causing a higher rate of conversion of dsb into chromatid aberrations.

Ataxia Telangiectasia↗

PubMed2002

Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair.

Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.

Acetylation↗

PubMed1999

Induced repair of DNA double-strand breaks at the G1/S-phase border.

Exposure of human cells to ionizing radiation at the G1/S-phase border of the cell cycle leads to the production of repair patches of 3 nucleotides, representing the constitutive repair response, and very long repair patches (VLRP) of at least 150 nucleotides, representing an induced response. We examined the type of DNA damage that may signal this induced repair response using two chemicals that produce subsets of the damage induced by ionizing radiation. Treatment of cells at the G1/S-phase border with bleomycin, which produces a high proportion of DNA double-strand breaks, also leads to the production of VLRP of at least 130 nucleotides. In contrast, when cells were treated with hydrogen peroxide, which produces base modifications and single-strand breaks, no VLRP were observed. Thus it would appear that DNA double-strand breaks are the signal that leads to the induction of the VLRP. We also examined the relationship between the induced repair response and DNA replication. When cells are treated with hydroxyurea, under conditions that inhibit more than 98% of the DNA synthesis, prior to exposure to 5 Gy, repair patches of 3 and 150 nucleotides are found. This indicates that the longer repair patches are not a result of aberrant DNA replication. However, when cells are treated with the DNA polymerase inhibitor aphidicolin in combination with hydroxyurea and cytosine arabinoside, no induced long patches are found. These results indicate that DNA polymerase alpha, delta or epsilon is required for the synthesis of the VLRP.

Aphidicolin↗

PubMed2005

DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons.

Linearised pUC 19 DNA with cohesive, blunt and non-matching ends, generated by prior treatment with different restriction enzymes was presented as substrate to measure the NHEJ activity to repair DNA double strand breaks in extracts prepared from isolated neurons from neonatal, young adult and old rat cerebral cortex. Highest end joining activity was noticed with the substrates having cohesive 3' overhang (PstI) or 5' overhang (EcoRI) ends and this activity is significantly reduced with age. However, blunt and non-matching ends were very poorly repaired at all ages. Further, the end joining activity in neurons is not faithful and sequence changes occur during the repair process. Also, the end joining activity in old neuronal extracts, but not in young extracts, was found to decline very rapidly with time of cold storage. These findings, the first of their kind, thus demonstrate that neuronal cells have the capacity to repair DNA double strand breaks through error prone NHEJ mode and that the cohesive end joining activity decreases with age of the animal.

Aging↗

PubMed1997

Chromosomal aberrations induced by defined DNA double-strand breaks: the origin of achromatic lesions.

The mechanisms of formation of chromosomal aberrations are poorly understood, despite the common use of aberrations as a measure of the genetic effects of physical and chemical agents. We have used restriction endonucleases to introduce defined DNA double-strand breaks into mammalian cells, and measured chromosomal aberration formation relative to the activity of the endonuclease. The endonucleases AluI and Sau3AI remain active for a relatively short time under simulated cellular conditions and induce achromatic lesions ('gaps') in chromatids only within the first hour or two following treatment. In contrast, the endonuclease MboI (an isoschizomer of Sau3AI) is active for an extremely long time and continues to produce chromatid gaps during the whole 12 hr sampling period. This observation strongly suggests that the aberrations classified as gaps are a manifestation of unrejoined DNA double-strand breaks. The formation of gaps may relate to the opportunities for repair of DNA breaks in relation to cell-cycle position. It is more difficult to relate the formation of structural chromatid aberrations to the endonuclease activity, although at relatively low concentrations all 3 endonucleases gave similar levels of structural aberrations.

Animals↗

PubMedDate not supplied

[Interaction of topotecan-a DNA topoisomerase I inhibitor-with dual-stranded polydeoxyribonucleotides. V. Topotecan is able to cause single- and double-strand breaks in ring superhelical DNA in the absence of enzyme].

Temperature, concentration, and time dependence for the emergence of breaks in the sugar-phosphate backbone in a circular supercoiled DNA (scDNA) was studied for the first time in the presence of topotecan (TPT) and in the absence of human DNA topoisomerase I (topo I). Because TPT is a comptothecin (CPT) derivative, it is the first example for the ability of molecules of CPT family to cause double-stranded breaks in scDNA in the absence of the enzyme. The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). Incubation time necessary for the appearance of double-stranded breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT-DNA complex. A model was suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of diverse strands of one of DNA duplexes. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines spaced five bp apart in the same strand of the second DNA duplex.

DNA Damage↗