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PubMed1992

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5.

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.

Animals↗

PubMed1997

Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7.

An in vitro DNA replication system based on extracts prepared from Escherichia coli cells infected with bacteriophage T7 was used to study deletion associated with the repair of double-strand breaks. The gene for T7 ligase was interrupted by a DNA insert which included 17-bp direct repeats. Deletion between the repeats restored the reading frame of the gene, and these DNA molecules could be detected by their ability to give rise to ligase-positive phage after in vitro packaging. T7 genomes that had a pre-existing double-strand break located between the direct repeats were incubated together with intact genomes which had the same direct repeats. Genetic markers placed on either side of the insert in the ligase gene allowed identification of the source of DNA molecules that underwent deletion between the direct repeats. This allowed an assessment of the participation of the molecules with strand breaks in the deletion process, under conditions where any mechanism could contribute to deletion. Approximately three-quarters of the T7 molecules that had lost the region between the direct repeats contained one or both of the partial genomes originally introduced into the reactions. About 50% of the genomes which had undergone deletion had recombined markers between the partial and intact genomes. The data demonstrate that double-strand breaks substantially enhance the contribution of intermolecular recombination to deletion.

Bacteriophage T7↗

PubMed2004

Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast.

The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA end-bridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging.

Adenosine Triphosphatases↗

PubMed1990

A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae.

We have identified and analyzed a meiotic reciprocal recombination hot spot in S. cerevisiae. We find that double-strand breaks occur at two specific sites associated with the hot spot and that occurrence of these breaks depends upon meiotic recombination functions RAD50 and SPO11. Furthermore, these breaks occur in a processed form in wild-type cells and in a discrete, unprocessed form in certain nonnull rad50 mutants, rad50S, which block meiotic prophase at an intermediate stage. The breaks observed in wild-type cells are similar to those identified independently at another recombination hot spot, ARG4. We show here that the breaks at ARG4 also occur in discrete form in rad50S mutants. Occurrence of breaks in rad50S mutants is also dependent upon SPO11 function. These observations provide additional evidence that double-strand breaks are a prominent feature of meiotic recombination in yeast. More importantly, these observations begin to define a pathway for the physical changes in DNA that lead to recombination and to define the roles of meiotic recombination functions in that pathway.

Blotting, Southern↗

PubMed1988

Verapamil reversal of doxorubicin resistance in multidrug-resistant human myeloma cells and association with drug accumulation and DNA damage.

Verapamil reversed resistance to doxorubicin in a human multiple myeloma cell line selected for multiple drug resistance. The drug-resistant cell line 8226/DOX40 is known to have reduced intracellular drug accumulation associated with the overexpression of P-glycoprotein when compared to the sensitive parent cell line 8226/S. Verapamil alone was minimally cytotoxic in both cell lines, but reversed doxorubicin resistance in a dose-related manner in 8226/DOX40. A similar dose-response relationship was observed for verapamil in increasing net intracellular doxorubicin accumulation. This increased net accumulation was secondary to block of enhanced doxorubicin efflux by verapamil from resistant cells. In contrast, verapamil did not alter initial doxorubicin accumulation over the first 60 s when incubated with resistant cells. Addition of verapamil to the 8226/DOX40 cells enhanced the formation of doxorubicin-induced DNA single strand breaks, double strand breaks, and DNA-protein cross-links. Verapamil had no effect on these lesions in the drug-sensitive cells. In addition, verapamil did not affect chemotherapeutic cytotoxicity or transport in the drug-sensitive cell line. Verapamil appears to reverse doxorubicin resistance in this human myeloma cell line by blocking enhanced drug efflux, leading to increased drug accumulation and enhanced DNA damage.

Cell Survival↗

PubMed2002

Induction of biphasic DNA double strand breaks and activation of multiple repair protein complexes by DNA topoisomerase I drug 7-ethyl-10-hydroxy-camptothecin.

Camptothecins demonstrate a broad spectrum of antitumor activity. Although they are known to trap DNA topoisomerase I on DNA, form cleavable complexes, and generate DNA breaks upon collision with DNA or RNA polymerases, the precise mechanisms predictive for antitumor activity remain to be identified. Recent studies using panels of colorectal and breast cancer cell lines indicate that events downstream of cleavable complexes are more relevant. In this study, we chose SN-38, an active metabolite of irinotecan, to characterize DNA double strand breaks and repair mechanisms induced by this type of drugs using a human head and neck squamous cell carcinoma cell line A253. The results showed that 2-h exposure of cells to an IC(50) concentration of SN-38 induces biphasic DNA double-strand break (DSBs): an immediate phase, which was greatly reduced within 8 h, and a lagging phase, culminating 24 h after drug removal. Three DNA double-strand break repair protein complexes were activated: DNA-dependent protein kinase (DNA-PK), NBS1-MRE11-RAD50, and BRCA1. Aphidicolin, a DNA polymerase inhibitor, abolished both phase I DSBs and the activation of repair protein complexes, suggesting that they resulted from the collision between the cleavable complex and DNA polymerase of S-phase cells. This is in contrast to ionizing radiation-induced activation of DNA-PK and NBS1-MRE11-RAD50 complexes that occur predominantly among non-S-phase cells. The trigger for phase II DSBs cannot be abolished by aphidicolin. The data also indicate that DNA fragments in the size of 50 to 200 kilobases were detected in the lagging phase. This suggests that the late DNA DSBs were associated with apoptotic cell death.

Aphidicolin↗

PubMedDate not supplied

Effects of the nucleoside analogues alpha-ara A, beta-ara A and beta-ara C on cell growth and repair of both potentially lethal damage and DNA double strand breaks in mammalian cells in culture.

The effects of the DNA polymerase inhibitors a-ara A and beta-ara C have been compared with those of beta-ara A on the endpoints of cell growth, repair of x-ray induced potentially lethal damage (PLD) and repair of x-ray induced DNA double strand breaks, a-ara A was found to have no effects on any of the endpoints studied. beta-ara C inhibited cell growth and DNA double strand breaks repair more strongly than beta-ara A but it was less effective in inhibiting repair of PLD. The inhibition of PLD repair by beta-ara C resulted in a diminution of the shoulder width of the x-ray survival curve, a result similar to that previously found for beta-ara A. The effectiveness of beta-ara C was enhanced when cells were treated with the drug in fresh medium rather than under plateau phase conditions. The lower effectiveness of beta-ara C, when compared with beta-ara A in causing expression of PLD, is interpreted in terms of a difference in the ability of the two drugs to cause fixation or misrepair of the DNA double strand breaks during or after treatment with the drugs.

Animals↗

PubMed2005

Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro.

The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

Animals↗

PubMed1975

Repair of double-strand breaks in Micrococcus radiodurans.

Micrococcus radiodurans has been shown to sustain double-strand breaks in its DNA after exposure to X-radiation. Following sublethal doses of X-rays (200 krad in oxygen or less), the cells were able to repair these breaks, and an intermediate fast-sedimenting DNA component seemed to be involved in the repair process.

Cell Membrane↗

PubMed1995

Detection of heavy-ion-induced DNA double-strand breaks using static-field gel electrophoresis.

Radiation-induced DNA double-strand breaks (DSBs) were measured in Chinese hamster ovary cells (CHO-K1) using an experimental protocol involving static-field gel electrophoresis following exposure to various accelerated ions. Dose-effect curves were set up, and relative biological efficiencies (RBEs) for DSB induction were determined for different radiation qualities. RBEs around 1 were obtained for low energy deuterons (6-7 keV/microns), while for high energy oxygen ions (20 keV/microns) an RBE value slightly greater than 1 was determined. Low energetic oxygen ions (LET approximately 250 keV/microns were found to show RBEs substantially below unity, and for higher LET particles (> or = 250 keV/microns) RBEs for DSB induction were generally found to be smaller than 1. The data presented here are in line with the generally accepted view that not induced DSBs, but rather misrepaired or unrepaired DNA lesions are related to cellular inactivation.

Animals↗

PubMed1990

Studies on mammalian mutants defective in rejoining double-strand breaks in DNA.

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.

Animals↗

PubMed1999

Locations of radiation-produced DNA double strand breaks along chromosomes: a stochastic cluster process formalism.

Ionizing radiation produces DNA double strand breaks (DSBs) in chromosomes. For densely ionizing radiation, the DSBs are not spaced randomly along a chromosome: recent data for size distributions of DNA fragments indicate break clustering on kbp-Mbp scales. Different DSB clusters on a chromosome are typically made by different, statistically independent, stochastically structured radiation tracks, and the average number of tracks involved can be small. We therefore model DSB positions along a chromosome as a stationary Poisson cluster process, i.e. a stochastic process consisting of secondary point processes whose locations are determined by a primary point process that is Poisson. Each secondary process represents a break cluster, typically consisting of 1-10 DSBs in a comparatively localized stochastic pattern determined by chromatin geometry and radiation track structure. Using this Poisson cluster process model, which we call the randomly located clusters (RLC) formalism, theorems are derived for how the DNA fragment-size distribution depends on radiation dose. The RLC dose-response relations become non-linear when the dose becomes so high that DSB clusters from different tracks overlap or adjoin closely. The RLC formalism generalizes previous models, fits current data adequately and facilitates mechanistically based extrapolations from high-dose experiments to the much lower doses of interest for most applications.

Chromosome Aberrations↗

PubMed2005

Characterization of complex apurinic/apyrimidinic-site clustering associated with an authentic site-specific radiation-induced DNA double-strand break.

Radiation lethality is largely attributed to radiation-induced DNA double-strand breaks (DSBs). A range of structural complexity is predicted for radiation-induced DSBs. However, this lesion has never been analyzed in isolation at the molecular level. To address this problem, we have created authentic site-specific radiation-induced DSBs in plasmid DNA by triplex-forming oligonucleotide-targeted 125I decay. No significant difference in DSB yield was observed after irradiation in the presence or absence of the radical scavenger DMSO, suggesting that DSB formation is a result of the direct effect of the radiation. A restriction fragment terminated by the DSB was isolated and probed with the Escherichia coli DNA repair enzyme endonuclease IV (endo IV), which recognizes apurinic/apyrimidinic (AP) sites. Enzymatic probing demonstrated clustering of AP sites within 10 bases of the 125I-targeted base in the DNA duplex. Our results suggest scavengeable radicals may not play a large role in the generation of AP sites associated with DSB formation, because at least 30% of all fragments have endo IV-sensitive sites, regardless of irradiation conditions. An internal control fragment recovered from the 125I linearized plasmid did not exhibit endo IV sensitivity in excess of that observed for a similar fragment recovered from an undamaged plasmid. Thus, AP site clustering proximal to the DSB resulted from the 125I decays responsible for DSB formation and was not due to untargeted background irradiation.

Base Sequence↗

PubMed1993

Lethality of heavy ion-induced DNA double-strand breaks in mammalian cells.

Cell killing and the production of DNA double-strand breaks (dsbs) were measured in parallel for V79 cells and a human tumour cell line (Caski) following exposure to radiations of differing linear energy transfer (LET) including accelerated heavy ions. Dsb induction was assessed by neutral filter elution (pH 7.4) and elution from agarose plugs in pulsed-field electrophoresis. The elution data were consistent with linear lesion induction and allowed to derive dsb yields or production cross-sections (sigma dsb), respectively. Both cell lines as well as the two elution approaches gave comparable results. sigma dsb was found to rise up to the heaviest ions used (LET = 11,500 keV/microns) but relative biological effectiveness was always smaller than unity. Slopes of the exponential survival curves (final slope with 60Co-radiation) were normalized to the respective dsb yields and allowed to calculate the relative lethality per induced dsb. This parameter increased with LET up to about 300 keV/microns followed by a steep decline for the very heavy ions. The extent of dsb rejoining was concomitantly reduced with ionization density.

Cell Death↗

PubMed1996

Dose-rate effect on radiation-induced DNA double-strand breaks in the human fibroblast HF19 cell line.

We measured DNA double-strand breaks (dsbs) immediately after exposure of a non-transformed human fibroblast cell line (HF19) to gamma-rays (0-40 Gy) at four dose-rates (10, 1, 0.1, and 0.01 Gy/min) at 37 degree C using clamped homogeneous electric field (CHEF) gel electrophoresis. The shape of the dose-response curves, which could be approximated by a straight line over the range 0-20 Gy for irradiation at 4 degree C, became curvilinear when irradiation was carried out at 37 degree C at 10, 1, 0.1, and 0.01 Gy/min and reached a plateau at 10 Gy after irradiation at 0.01 Gy/min. We present a mathematical analysis that predicts the results of irradiation at 37 degree C from dsb induction and repair data obtained at 4 degree C, followed by incubation for repair at 37 degree C. The model assumes that the rate of dsb rejoining changes continuously with repair time and that it is independent of dose and dose-rate in the range 10-40 Gy. The model also assumes a linear induction of dsb with dose at 4 degree C and dsb induction is independent of dose-rate and of temperature during irradiation. Independent measurements of dsb induction at 4 degree C and of repair rate accurately predict the dsb levels after irradiation at 37 degree C, during which both phenomena occur simultaneously.

DNA↗

PubMed1998

Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.

The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.

Adenosine Triphosphatases↗

PubMed1979

Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons).

Animals↗

PubMed1992

Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1.

Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3' ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.

DNA Repair↗