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PubMed2001

Requirement for repair of DNA double-strand breaks by homologous recombination in split-dose recovery.

Utsumi, H., Tano, K., Takata, M., Takeda, S. and Elkind, M. M. Requirement for Repair of DNA Double-Strand Breaks by Homologous Recombination in Split-Dose Recovery. Radiat. Res. 155, 680-686 (2001). Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and is believed to be the result of the repair of sublethal damage. It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism(s) of this repair, we analyzed the knockout mutants KU70-/-, RAD54-/-, and KU70-/-/RAD54-/- of the chicken B-cell line, DT40. RAD54 participates in the recombinational repair of DNA double-strand breaks (DSBs), while members of the KU family of proteins are involved in nonhomologous end joining. Split-dose recovery was observed in the parent DT40 and the KU70-/- cells. Moreover, the split-dose survival enhancement had all of the characteristics demonstrated earlier for the repair of sublethal damage, e.g., the reappearance of the shoulder on the survival curve with dose fractionation; cyclic fluctuation in cell survival at 37 degrees C; repair and no cyclic fluctuation at 25 degrees C. These results strongly suggest that repair of sublethal damage is due to DSB repair mediated by homologous recombination, and that these DNA DSBs constitute sublethal damage.

Animals↗

PubMed2000

Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting.

In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.

Base Sequence↗

PubMed1991

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation.

The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the KpnI restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung+ but not in extracts from ung- E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet+ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung+ E. coli strain in comparison with an ung- strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.

Base Sequence↗

PubMed1991

Analysis of restriction enzyme-induced DNA double-strand breaks in Chinese hamster ovary cells by pulsed-field gel electrophoresis: implications for chromosome damage.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.

Animals↗

PubMed2000

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Base Sequence↗

PubMed1998

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.

Adult↗

PubMed2002

Conversion of phosphoglycolate to phosphate termini on 3' overhangs of DNA double strand breaks by the human tyrosyl-DNA phosphodiesterase hTdp1.

Mammalian cells contain potent activity for removal of 3'-phosphoglycolates from single-stranded oligomers and from 3' overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3' terminus to leave a 3'-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3'-phosphoglycolate on a 3-base 3' overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3' overhangs.

Base Sequence↗

PubMed2002

Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair.

The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.

Acetyltransferases↗

PubMed2006

Quantitative measurement of hydroxyl radical induced DNA double-strand breaks and the effect of N-acetyl-L-cysteine.

Reactive oxygen species, such as hydroxyl or superoxide radicals, can be generated by exogenous agents as well as from normal cellular metabolism. Those radicals are known to induce various lesions in DNA, including strand breaks and base modifications. These lesions have been implicated in a variety of diseases such as cancer, arteriosclerosis, arthritis, neurodegenerative disorders and others. To assess these oxidative DNA damages and to evaluate the effects of the antioxidant N-acetyl-L-cysteine (NAC), atomic force microscopy (AFM) was used to image DNA molecules exposed to hydroxyl radicals generated via Fenton chemistry. AFM images showed that the circular DNA molecules became linear after incubation with hydroxyl radicals, indicating the development of double-strand breaks. The occurrence of the double-strand breaks was found to depend on the concentration of the hydroxyl radicals and the duration of the reaction. Under the conditions of the experiments, NAC was found to exacerbate the free radical-induced DNA damage.

Acetylcysteine↗

PubMed2001

Autoantibodies against DNA double-strand break repair proteins.

Autoantibodies against cellular components are commonly present in sera from patients with systemic rheumatic diseases and may play an important role in pathogenesis. The Ku protein was recognized 20 years ago as a major target of autoantibodies in a subset of Japanese patients with scleroderma-polymyositis overlap syndrome, and anti-Ku antibodies have since been shown to occur in 10-20% of patients with these and other systemic rheumatic diseases, including systemic lupus erythematosus. Ku functions physiologically in the repair of DNA double-strand breaks, where it carries out the initial recognition of damaged DNA ends. The three dimensional structure of the Ku-DNA complex has recently been solved, and helps illuminate the relationship between the autoimmune epitopes and other features of the protein. In addition to Ku, three other polypeptides in the same DNA repair pathway have more recently been identified as autoantigens: the DNA-dependent protein kinase catalytic subunit, DNA ligase IV, and XRCC4. Two hypotheses have been invoked to explain the ability of these proteins to elicit an autoimmune response in susceptible individuals. One is that DNA damage induces formation of nucleoprotein complexes that present novel composite or conformational epitopes. The other is that cleavage of these proteins by caspases or Granzyme B leads to presentation of immunocryptic peptides capable of stimulating autoreactive T lymphocytes. In the case of DNA double-strand break repair proteins, there is evidence that both of these mechanisms may be at work. Because of their role in the maintenance of genome stability, DNA double-strand break repair proteins have been the subject of intense study, and a wealth of new structural, biochemical and functional information makes them excellent models for investigation of the humoral autoimmune response.

Animals↗

PubMed1998

Implication of DNA-dependent protein kinase in an early, essential, local phosphorylation event during end-joining of DNA double-strand breaks in vitro.

Previous work with Xenopus egg extracts suggested that a wortmannin-sensitive protein phosphorylation event precedes both the removal of modified termini from DNA double-strand break ends and the joining of unmodified ends. To assess the possible role of DNA-dependent protein kinase in effecting this phosphorylation, both DNA end-joining and DNA-stimulated phosphorylation were examined in the presence of various inhibitors. Linear but not supercoiled DNA stimulated the phosphorylation of several endogenous proteins in the extracts, including species of approximately 48, 87, and 96 kDa. This phosphorylation was selectively suppressed by the kinase inhibitors wortmannin, dimethylaminopurine, and LY294002, with a dose response that in each case paralleled the inhibition of DNA end-joining. If wortmannin was added while the end-joining reaction was in progress, end-joining of DNA already present in the reaction continued for some time, but newly added DNA was not joined or processed at all. Ends with 3'-hydroxyl termini were joined much faster than those with 3'-phosphoglycolate termini, although both were equally effective in stimulating protein phosphorylation. The results support a role for DNA-dependent protein kinase in regulating end-joining in vitro, and suggest that at least one of the necessary phosphorylations involves a protein bound at or near the DNA end to be joined. In contrast, nuclear extracts from human cells joined double-strand breaks with normal but not modified termini, and the joining was unaffected by kinase inhibitors, suggesting that the dominant mechanism of end-joining in these extracts did not involve DNA-PK.

Amino Acid Sequence↗

PubMed1998

Investigation of the induction of DNA double-strand breaks by methylenediphenyl-4-4'-diisocyanate in cultured human lung epithelial cells.

The question was addressed whether methylenediphenyl-4,4'-diisocyanate (MDI), a bifunctional electrophile, can induce DNA double-strand breaks (DSB) by repair of interstrand DNA crosslinks or whether DSB are the result of cell death. Cultured human lung epithelial cells (A549) were treated with MDI, methylene-4,4'-dianiline (MDA; a potential hydrolysis product of MDI), the nitrogen mustard melphalan, and the detergent Triton X-100. All chemicals were dissolved in ethylene glycol dimethyl ether which was added to a cell monolayer covered with phosphate-buffered saline. After 2 h, the treatment solution was exchanged against medium, and 8, 24, and 72 h after treatment initiation, the induction of DNA double-strand breaks was assessed by pulsed-field gel electrophoresis. At the same time, the viability was determined with the MTT test (intracellular reduction of the tetrazolium dye MTT). At the 8-h time point, 1 and 10 microM melphalan induced DSB without concomitant effect on cell viability. With all other chemicals, the dose-response curves for DNA fragmentation and viability were mirror images. Approximate 50% lethal concentrations were 200, 3000, and 100 microM for MDI, MDA, and Triton X-100, respectively. For these chemicals, the observed DSB were the consequence of extragenomic damage in the course of cell death rather than of an interaction with DNA. The mechanistic difference of melphalan was supported by analysis of nuclear morphology. Apoptotic bodies were observed only after melphalan treatment, whereas MDI and Triton X-100 produced only irregular clumping of chromatin (72-h time point). DNA fragment length analysis showed a time-independent pattern, with sizes between 1 and 4 Mbp for melphalan, while MDI, and Triton X-100 induced smaller DNA fragments in a time-dependent manner. It is concluded that DSB observed in cells treated with MDI are unlikely the result of DNA crosslink formation.

Aniline Compounds↗

PubMed2001

Asp-Ala-His-Lys (DAHK) inhibits copper-induced oxidative DNA double strand breaks and telomere shortening.

Both DNA and the telomeric sequence are susceptible to copper-mediated reactive oxygen species (ROS) damage, particularly damage attributed to hydroxyl radicals. In this study, ROS-induced DNA double strand breaks and telomere shortening were produced by exposure to copper and ascorbic acid. Asp-Ala-His-Lys (DAHK), a specific copper chelating tetrapeptide d-analog of the N-terminus of human albumin, attenuated DNA strand breaks in a dose dependent manner. d-DAHK, at a ratio of 4:1 (d-DAHKCu), provided complete protection of isolated DNA from double strand breaks and, at a ratio of 2:1 (d-DAHKCu), completely protected DNA in Raji cells exposed to copper/ascorbate. Southern blots of DNA treated with copper/ascorbate showed severe depletion and shortening of telomeres and Raji cell treated samples showed some conservation of telomere sequences. d-DAHK provided complete telomere length protection at a ratio of 2:1 (d-DAHKCu). The human albumin N-terminus analog, d-DAHK, protects DNA and telomeres against copper-mediated ROS damage and may be a useful therapeutic adjunct in ROS disease processes.

Cell Line↗

PubMed1975

Comparsion of the oxygen-enhancement ratio for gamma-ray-induced double-strand breaks in the DNA of bacteriophage T7 as determined by two different methods of analysis.

Bacteriophage T7 was irradiated in a protecting medium under nitrogen and oxygen with 60Co gamma-rays. Double-strand breaks were measured by sucrose-gradient sedimentation and by boundary-sedimentation analysis. Both methods showed that the presence of oxygen during irradiation enhanced the production of double-strand breaks. This is at variance with a recent report which suggests that boundary-sedimentation analysis does not show an effect of oxygen. The discrepancy must be ascribed to differences in interpretation of the sedimentation data.

Centrifugation, Density Gradient↗

PubMed2026

Efficient and precise programmable DNA knock-in without double-strand breaks.

Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

Animals↗

PubMed1990

Detection of ionizing radiation-induced DNA double-strand breaks by filter elution is affected by nuclear chromatin structure.

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.

Animals↗

PubMed1995

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae.

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 bp deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 bp deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.

Chromosomes, Fungal↗

PubMed2006

Fission yeast Cut8 is required for the repair of DNA double-strand breaks, ribosomal DNA maintenance, and cell survival in the absence of Rqh1 helicase.

Schizosaccharomyces pombe Rqh1 is a member of the RecQ DNA helicase family. Members of this protein family are mutated in cancer predisposition diseases, causing Bloom's, Werner, and Rothmund-Thomson syndromes. Rqh1 forms a complex with topoisomerase III and is proposed to process or disrupt aberrant recombination structures that arise during S phase to allow proper chromosome segregation during mitosis. Intriguingly, in the absence of Rqh1, processing of these structures appears to be dependent on Rad3 (human ATR) in a manner that is distinct from its role in checkpoint control. Here, we show that rad3 rqh1 mutants are normally committed to a lethal pathway of DNA repair requiring homologous recombination, but blocking this pathway by Rhp51 inactivation restores viability. Remarkably, viability is also restored by overexpression of Cut8, a nuclear envelope protein involved in tethering and proper function of the proteasome. In keeping with a recently described function of the proteasome in the repair of DNA double-strand breaks, we found that Cut8 is also required for DNA double-strand break repair and is essential for proper chromosome segregation in the absence of Rqh1, suggesting that these proteins might function in a common pathway in homologous recombination repair to ensure accurate nuclear division in S. pombe.

Cell Cycle Proteins↗