DNA double-strand breaks in mutagenesis.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
W F Morgan
Publications and source records attributed to W F Morgan.
Telomeres and their possible role in chromosome stabilization.
The evidence to date generally supports the hypothesis that telomere capping makes chromosome fragments refractory to subsequent rejoining events, but this control may be somewhat relaxed after chromosome breakage. Cell survival requires that the fragments rejoin before metaphase. Unprotected ends such as those produced by DNA damage are subject to degradation, presumably by endogenous cellular exo- and endonucleases. Telomere repeat sequences may be added to broken chromosome ends to protect the ends from further degradation. That telomeric DNA does not always prevent rejoining raises interesting questions as to what constitutes capping, and how rapidly it occurs after DNA damage in relation to chromosome break rejoining. The prevention of degradation and control of rejoining may be mediated by telomere-specific binding proteins, especially the telomere terminal binding protein [Gualberto et al., 1992; Longtine et al., 1989; Price, 1990; Price and Cech, 1989]. Some of these proteins may be involved in scavenging telomeric DNA when the cell senses that chromosomal breaks have occurred. This mechanism is consistent with the observations of Murnane and Yu [1993], who found that a plasmid with telomere sequences was stably integrated in vivo into a chromosome terminal breakpoint lacking telomere repeats. It is also consistent with the high frequency of interstitial telomere sequences observed in normal cells; a history of DNA damage and repair may be recorded by these sequences (Ijdo et al., 1991]. Although chromosome break rejoining is an efficient process in eukaryotic cells, some breaks are never rejoined and can result in terminal deletions and chromatid and isochromatid deletions at metaphase. It is unclear why these breaks are not rejoined, but it may be due to one or more of the following: 1) chance: broken chromosomes are separated, do not approach sufficiently close to one another, and are consequently physically unable to rejoin; 2) a large number of added telomere repeat sequences indicating to the cell that the chromosome has an authentic telomere; 3) some other DNA modification event that protects DNA ends from degradation, e.g., folding back of DNA ends to form a hairpin, as has been implicated in VDJ recombination [Lieber, 1993].
Animals↗
Nonhomologous DNA end rejoining in chromosomal aberration formation.
The role of recombination of nonhomologous DNA ends in chromosomal aberration formation was investigated in Chinese hamster ovary cells. Restriction enzymes that produce blunt, 3' overhanging, or 5' overhanging DNA double-strand breaks were electroporated into cells in various combinations, and chromosomal aberrations were analyzed at metaphase. For all enzyme combinations tested, there was a significant increase in the frequency of aberrations whose formation requires two breaks in the DNA over the sum obtained when each of the enzymes was tested separately and the aberration frequencies were totaled. No such pattern existed for terminal deletions, which presumably require only one DNA break. The extent of interaction did not depend on the homology in the overhanging sequences or on the combination of ends used, although the largest effect was seen with a combination of two blunt ends. This study shows that nonhomologous DNA double-strand breaks can interact to increase chromosomal aberration formation significantly.
Animals↗
Mechanisms involved in rejoining DNA double-strand breaks induced by ionizing radiation and restriction enzymes.
DNA double-strand breaks are considered to be the most deleterious lesion induced by ionizing radiation. However, the mechanism of rejoining of these lesions has not been extensively studied at the molecular level. We have used a shuttle vector, pHAZE, to analyze the mechanism of rejoining of DNA double-strand breaks in human cells. The advantage of this vector system is that, unlike many previously described shuttle vectors, it has a large target gene for the detection of deletions and it is maintained as a freely replicating episome with chromatin conformation in the nucleus of human cells. In this study we compare data obtained on the spectrum of mutations induced in pHAZE by ionizing radiation (alpha-particles) and restriction enzymes (PvuII, ClaI, and PvuI). Unlike ionizing radiation, restriction enzymes induce double-strand breaks in DNA with known end structures at defined locations and therefore provide a model system for analyzing cellular responses to DNA double-strand breaks. Exposure of human cells containing the vector to alpha-particle irradiation produced both point mutations and large deletions in pHAZE. When the junction regions of the deletions were sequenced it was found that 65% were rejoined with up to 6 bp of homology at the junction region. Analysis of restriction-enzyme-induced mutations suggests that double-strand break ends are modified to facilitate rejoining and that the type of modification is characteristic for different end structures. Double-strand breaks with cohesive ends appear to have fewer modifications introduced at the break points before rejoining than breaks with blunt ends. When considered in relation to the data obtained with ionizing radiation this suggests that the presence of cohesive sequences either at, or in proximity to, the ends enhances rejoining of DNA double-strand breaks.
Base Sequence↗
Gonadotrophin-releasing hormone treatment in cattle: a meta-analysis of the effects on conception at the time of insemination.
Data from 40 trials described in 27 published papers were analysed by meta-analysis (pooling of data from numerous trials into a single analysis) to evaluate the effects of injecting gonadotrophin-releasing hormone (GnRH) in cattle at the time of insemination on the risk of pregnancy. A total of 19,019 cows were involved in these studies. Analyses were stratified by trial and by the effects of dose of GnRH, type of GnRH and insemination number (first, second or third and greater). Mantel-Haenszel analysis revealed that use of GnRH at insemination significantly increased the overall risk of pregnancy by 12.5% in treated cows (P < 0.05). However, increases in risk of pregnancy were greatest (22.5%) when repeat breeders were tested. Results for use of GnRH or analogue at first service were similar, with increased risks of pregnancy in treated cows of 5.2 and 8.0%, respectively. The risk of pregnancy tended to be higher (11.1%) when the dose of GnRH was > or = 250 micrograms and when used at second insemination (9.9%). The analyses demonstrated that while treatment at insemination with GnRH and GnRH analogues increased conception rates in dairy cattle, some variation in study results was attributable to the number of inseminations after calving at which GnRH is administered. Further efforts should be made to determine characteristics of populations of cows that have good fertility responses to GnRH and to determine the mode of action of GnRH in increasing fertility.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Delayed chromosomal instability induced by DNA damage.
DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability.
Animals↗
Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.
Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.
Benzamides↗
Restriction enzyme-induced DNA double-strand breaks as a model system for cellular responses to DNA damage.
To learn more about cellular responses to DNA double-strand breakage, we used three methods to assay cellular damage after treatment with a restriction enzyme that causes DNA double-strand breaks by cleaving at specific recognition sites in the DNA. Chinese hamster ovary cells were treated with increasing doses of Pvu II and studied for double-strand breakage, chromosomal aberration yield, and cell survival. The yield of DNA double-strand breaks, as measured by pulsed-field gel electrophoresis, increased at concentrations up to 500 units and saturated thereafter. The maximum yield of metaphase cells showing aberrant chromosomes was reached at 100 units and stayed constant up to 1,000 units. Although exchange-type aberrations saturated at approximately 4.5 per cell at 100 units, deletion-type aberrations appeared to increase at concentrations up to 500 units. Cell survival, as measured by colony-forming ability after Pvu II treatment, saturated at 100 units. The observed dose-response data are probably due to the saturation of accessible Pvu II cleavage sites within the cell. These data indicate that restriction enzymes induce the same DNA-damaging effects as many of the agents used in cancer treatment. Because the primary DNA lesion induced by restriction enzymes is known, they provide a unique opportunity to understand cellular responses to DNA damage and repair.
Animals↗
Modelling the formation of polycentric chromosome aberrations.
Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.
Animals↗
Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks.
Rejoining of DNA double-strand breaks (DSB) plays a central role in the various processes leading to DNA rearrangements. We have analyzed DNA alterations induced by restriction enzymes that produce DSB with specific types of ends. Restriction enzymes were electroporated into a human lymphoblastoid cell line that stably maintains pHAZE, an EBV-based vector containing the lacZ gene. After allowing time for DSB repair, pHAZE DNA was rescued and screened in Escherichia coli. Mapping and sequence analysis of mutant copies of pHAZE indicated that restriction enzymes induced all classes of alterations except base substitutions (base deletions and insertions, large-scale deletions, inversions, and insertions). The spectra of alterations were distinctive for each enzyme and appear to be the consequence of specific end-modification processes.
Base Sequence↗
Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double-strand breaks alone.
Malignant transformation of mouse fibroblasts was produced by electroporation with restriction enzymes. Similar transformation frequencies were observed with Pstl, Pvull, and Xbal, which cut genomic DNA at similar overall frequencies but have different termini, i.e., a 3' overhang, a blunt end, and a 5' overhang, respectively. The dose-response curve for restriction enzyme transformation shows a marked plateau in frequencies of transformed foci per surviving cell, whereas x-irradiation of the same cells gives a linear dose-response curve. Evidently, transformation can be caused by DNA double-strand breaks alone at a limited number of sites, but the evidence from x rays suggests that other kinds of DNA damage can cause transformation independently.
Animals↗
Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation.
The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.
Bromodeoxyuridine↗
Poly(ADP-ribose)polymerase: a perplexing participant in cellular responses to DNA breakage.
Poly(ADP-ribose) polymerase is a major nuclear protein of 116 kd, coded by a gene on chromosome 1, that plays a role in cellular responses to DNA breakage. The polymerase binds to DNA at single- and double-strand breaks and synthesizes long branched chains of poly(ADP-ribose), which covalently, but transiently, modifies itself and numerous other cellular proteins and depletes cells of NAD+. This much is known, but the physiological role of the polymerization-degradation cycle is still unclear. Poly(ADP-ribosyl)ation of proteins generally inhibits their function and can dissociated chromatin proteins from DNA. Inhibition of poly(ADP-ribose) polymerase increases to toxicity of alkylating agents and some other DNA-damaging agents and increases sister-chromatid exchange frequencies. During repair of alkylation damage, inhibition of poly(ADP-ribose) polymerase makes no change in excision of damaged products. increases the total number of repair patches, accelerates the rejoining of DNA breaks, and makes variable increases or decreases in net break frequencies. The polymerization cycle consequently is a major player in the response of cells to DNA breakage, but the game it plays is yet to be explained.
Amino Acid Sequence↗
Chromosomal aberration induction in CHO cells by combined exposure to restriction enzymes and X-rays.
The potential interaction between restriction enzyme-induced double-strand breaks (dsb) and X-ray-induced lesions in the formation of chromosomal aberrations was investigated in Chinese hamster ovary cells. Either Alu I, which induces blunt-end dsb, or Sau 3AI, which induces cohesive-end dsb, was electroporated into cells, which were irradiated with 2 Gy of X-rays immediately or 15, 30, 60, 120, or 180 min after electroporation. A significant increase in Alu I-induced chromosomal aberrations was observed when cells were irradiated with 0, 15, 30, or 60 min after enzyme exposure, but only additive effects were found when cells were irradiated 120 or 180 min after enzyme exposure. In one of three experiments, cells exposed to Sau 3AI showed a large increase in aberrations when X-irradiated 0 or 15 min after Sau 3AI exposure, and no increase at any time-points thereafter. These results indicate that restriction enzyme-induced dsb can interact with X-ray-induced lesions, resulting in a synergistic increase in chromosomal aberration formation. Furthermore, this interaction depends on both the type of dsb and the time between enzyme and X-ray exposure.
Animals↗
Induction of chromosome damage by restriction enzymes during mitosis.
Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.
Animals↗
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↗
Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis.
The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.
Animals↗
X-ray induction of methotrexate resistance due to dhfr gene amplification.
The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.
Animals↗