The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.     

Duplex-duplex interactions catalyzed by recA protein allow strand exchanges to pass double-strand breaks in DNA

Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E. coli RecA protein promotes strand exchanges past double-strand breaks. The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro. These results show that RecA protein can promote pairing interactions between homologous DNA molecules at regions where both are duplex. Moreover, pairing leads to strand exchanges and the formation of heteroduplex DNA. In contrast, strand exchanges are unable to pass a double-strand break in the gapped substrate. This apparent paradox is discussed in terms of a model for RecA-DNA interactions in which we propose that each RecA monomer contains two nonequivalent DNA-binding sites.     

The formation of UV-induced chromosome aberrations involves ERCC1 and XPF but not other nucleotide excision repair genes

ERCC1-XPF, through its role in nucleotide excision repair (NER), is essential for the repair of DNA damage caused by UV light. ERCC1-XPF is also involved in recombinational repair processes distinct from NER. In rodent cells chromosome aberrations are a common consequence of UV irradiation. We have previously shown that ERCC1-deficient cells have a lower ratio of chromatid exchanges to breaks than wild type cells. We have now confirmed this result and have shown that XPF-deficient cells also have a lower ratio than wild type. However, cells deficient in the other NER genes, XPD, XPB and XPG, all have the same ratio of exchanges to breaks as wild type. This implies that ERCC1-XPF, but not other NER proteins, is involved in the formation of UV-induced chromosome aberrations, presumably through the role of ERCC1-XPF in recombinational repair pathways rather than NER. We suggest that ERCC1-XPF may be involved in the bypass/repair of DNA damage in replicating DNA by an exchange mechanism involving single strand annealing between non-homologous chromosomes. This mechanism would rely on the ability of ERCC1-XPF to trim non-homologous 3' tails.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VII. Complementation analysis of X-irradiated wild-type CHO-K1 and xrs mutant cells using the premature chromosome condensation technique

The complementation effect of wild-type CHO-K1 and xrs mutants after fusion, as judged by the frequencies of X-ray-induced G1 and G2 premature chromosome condensation (PCC), was studied. For induction of PCC, X-irradiated interphase cells (G1 and G2) were fused immediately with untreated mitotic cells of the same cell line or with mitotic cells of another line. The frequencies of breaks in G1-PCC, or breaks and chromatid exchanges in G2-PCC were determined and the latter parameter was compared with the frequency of chromosomal aberrations in mitotic cells following G2 irradiation. CHO-K1 cells were capable of complementing the X-ray sensitivity of both xrs 5 and xrs 6 cells. However, full restoration of the repair defect in xrs cells could never be accomplished. The mutants failed to complement each other. In CHO-K1 cells, the incidence of chromosomal aberrations was significantly higher in G2-PCC (2.5-fold) than that observed in mitotic cells at 2.5 h after irradiation. The ratio of the induced frequency of aberrations in G2-PCC to that in mitotic cells was correlated with the degree of repair of DNA double-strand breaks (dsb) and reached almost 1 in xrs 5 cells indicating no repair. In addition the data indicated that, during the period of recovery of CHO-K1 cells, X-ray-induced breaks decreased but exchanges remained at the same level. In contrast, due to a deficiency in rejoining of dsb in xrs mutants, breaks remained open for a long period of time, allowing the formation of additional chromatid exchanges during recovery time.     

Possible correlations between cell killing, chromosome damage and DNA repair after X-irradiation

To gain information about the possible pathway from primary DNA damage to cell killing via the formation of chromosome aberrations, we have examined the effects of the DNA synthesis inhibitor ara A on survival, on the occurrence of chromosome abnormalities and on the repair of DNA strand breaks. Our results are not inconsistent with the idea that the increased expression or 'fixation' of PLD measured after treatment with ara A is a reflection of an increase in the formation of chromosome damage comprising both exchange type and deletion type aberrations. These aberrations may arise from unrepaired or misrepaired dsb in the DNA. Treatment of irradiated cells with ara A results in a larger number of residual dsb which may be partly the reason for the increase in the frequency of acentric chromosome fragments. The reasons for the increase also in the frequency of exchange aberrations in the presence of ara A are not known but one possibility is that the probability of interaction between two dsb remains high during treatment with ara A due to the strong inhibition of dsb repair, whereas in untreated controls this probability decreases steeply with time after irradiation.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein

The unresolved mechanism by which a single strand of DNA recognizes homology in duplex DNA is central to understanding genetic recombination and repair of double-strand breaks. Using stopped-flow fluorescence we monitored strand exchange catalyzed by E. coli RecA protein, measuring simultaneously the rate of exchange of A:T base pairs and the rates of formation and dissociation of the three-stranded intermediates called synaptic complexes. The rate of exchange of A:T base pairs was indistinguishable from the rate of formation of synaptic complexes, whereas the rate of displacement of a single strand from complexes was five to ten times slower. This physical evidence shows that a subset of bases exchanges at a rate that is fast enough to account for recognition of homology. Together, several studies suggest that a mechanism governed by the dynamic structure of DNA and catalyzed by diverse enzymes underlies both recognition of homology and initiation of strand exchange.     

Evolution of DNA damage in irradiated cells

Ionizing radiation damage to a mammalian genome is modeled using continuous time Markov chains. Models are given for the initial infliction of DNA double strand breaks by radiation and for the enzymatic processing of this initial damage. Damage processing pathways include DNA double strand break repair and chromosome exchanges. Linear, saturable, or inducible repair is considered, competing kinetically with pairwise interactions of the DNA double strand breaks. As endpoints, both chromosome aberrations and the inability of cells to form clones are analyzed. For the post-irradiation behavior, using the discrete time Markov chain embedded at transitions gives the ultimate distribution of damage more simply than does integrating the Kolmogorov forward equations. In a representative special case explicit expressions for the probability distribution of damage at large times are given in the form used for numerical computations and comparisons with experiments on human lymphocytes. A principle of branching ratios, that late assays can only measure appropriate ratios of repair and interaction functions, not the functions themselves, is derived and discussed.This work was supported in # DMS-9025103     

Repair of DNA single-strand breaks in X-irradiated yeast. II. Kinetics of repair as measured by the DNA-unwinding method

The kinetics of disappearance of single-strand breaks (SSB) from the DNA of X-irradiated stationary yeast cells under liquid-holding conditions was found to proceed in a dose-independent manner up to a dose of at least 2400 Gy, and was found to be complete after incubation of cells for 1 h. This was deduced from data for a yeast wild-type (WT) haploid and diploid strain as well as for rad52 haploid cells defective in DNA double-strand break (DSB) repair. In all cases an initial fast repair component assumed to correspond to SSB repair was observed whereby about 80% of the induced 'unwinding points' disappeared from the DNA with a time constant of about 3 min. Following this fast component, a slower component of removal of 'unwinding points' occurred with a time constant estimated to be 20 min. The molecular nature of these two components of repair is not known. We could find no evidence for the induction of secondary (enzymatic) breaks in the DNA during post-irradiation incubation. Incubation of cells in growth medium after irradiation resulted in similar kinetics as those under liquid-holding conditions. In the absence of an energy source in the medium (i.e. when cells were incubated in buffer or distilled water after irradiation) only 60-80% of the SSB were removed from yeast DNA. Residual SSB disappeared from the DNA only when cells were transferred to a medium containing glucose. The relative mass of DNA unwound per induced strand break (i.e. represented by the slope of the dose-effect curve immediately after irradiation) was found to change slowly with the age of the cell culture under liquid-holding conditions. This effect had to be corrected for in the measurements of strand break repair under these conditions.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.     

Disorders of repair processes in the lymphocytes of schizophrenia patients, cultured in vitro

Repair disorders of DNA damage induced by gamma-radiation and 4-nitroquinoline-1-oxide treatment in cultivated lymphocytes of patients with schizophrenia. 13 criteria were used for estimation of repair activity (reactivation of viral host cells) repair synthesis, reparation of DNA breaks, formation of spontaneous and induced sister chromatid exchanges.     

The ratio of dicentrics to centric rings produced in human lymphocytes by acute low-LET radiation.

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise, in an exchange event. Dicentrics and centric rings are aberrations that exemplify inter- and intrachromosomal exchanges, respectively. We show from a survey of published data that for acute low-LET irradiation of resting human lymphocytes the observed ratio of dicentrics to centric rings is approximately five times smaller than predicted by a pairwise interaction model which assumes complete randomness. Such a low ratio can be interpreted as evidence for a proximity effect, favoring exchanges of an intrachromosomal type. That is, since DSBs induced close together have an above-average chance of pairwise interaction, the observed excess of centric rings indicates that at the time of irradiation there is some degree of spatial confinement for the two arms of a single chromosome. Assuming the excess of centric rings is indeed due to proximity effects, the data are used to estimate that the volume of a domain, within which any one lymphocyte chromosome is localized at one instant during the G0/G1 phase, is at most approximately 20% of the nuclear volume.     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

The recovery parameters of DNA strand breaks of Ehrlich ascites cells after the combined action of ionizing radiation and hyperthermia

A mathematical model of DNA strand breaks postirradiation repair and the methodology allowing to differentiate the mechanism of inhibition of DNA strand breaks recovery after combined actions of ionizing radiation and hyperthermia have been described in this paper. Using this model and the results published by other authors for DNA strand breaks of Ehrlich ascites cells, there have been obtained the data showing that the portion of DNA-damages that the cell incapable to recover after consecutive thermoradiation action was risen with an increase in thermal load under insignificant change of repair constant. It means the mechanism of DNA strand breaks recovery inhibition is realized in a greater extent through the formation of irreversible damages but not through the damage of repair process itself.     

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined.Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.     

The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation

Strand breaks were detected in the DNA of Ehrlich ascites cells as well as in HeLa S3 cells directly after 1-5 hr at 43-45 degrees C by the use of the unwinding in high salt/hydroxylapatite method. The strand breaks found could not be attributed to the decay of incorporated tritiated thymidine. When the cells were incubated at 37 degrees C after the hyperthermic treatments, the amount of strand breaks formed remained at a constant level. Hyperthermia inhibited the repair of "radiation-induced" strand breaks. The repair curves obtained this way show a heat-dose-dependent decrease of the relative weight of the fast component of repair. Similar repair curves of "radiation-induced" strand breaks could be obtained by mixing heat inactivated and vital control cells prior to irradiation. In the latter case, however, the DNA repair was inhibited to a greater extent for identical levels of cell survival. The possible underlying molecular mechanisms are discussed.     

Abnormal sister-chromatid exchange induction by 3-aminobenzamide in an SV40-transformed Indian muntjac cell line: relationships with DNA maturation and DNA-strand breakage.

In SVM cells, an SV40-transformed line of Indian muntjac fibroblasts, levels of sister-chromatid exchanges are known to be abnormally high after UV-irradiation or alkylation. The SVM line is also known to have a defect in the processing of DNA-strand breaks. Sister-chromatid exchange in other cells is known to be stimulated by the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide, which also retards DNA-break sealing. Sister-chromatid exchanges in SVM cells are found to be hypersensitive to 3-aminobenzamide, or to nicotinamide deprivation which similarly inhibits poly(ADP-ribosyl)ation; DNA-strand breaks are likewise induced by 3-aminobenzamide. Bromodeoxyuridine, needed to detect sister-chromatid exchanges, is more toxic to SVM cells and itself induces sister-chromatid exchanges to a greater extent than it does in normal muntjac cells. However, in contrast to the situation reported for other cell types prone to sister-chromatid exchange (the Chinese hamster ovary mutant EM9 and human Bloom's Syndrome cells), SVM cells do not show an abnormal delay in DNA maturation when, under the influence of bromodeoxyuridine and 3-aminobenzamide, they show a high level of sister-chromatid exchange. The mechanism by which BrdU exerts its effects can largely be explained in terms of familiar effects on deoxyribonucleotide pools and DNA integrity. 3-Aminobenzamide, however, induces sister-chromatid exchanges in SVM cells by another mechanism.     

A replication model for sister-chromatid exchange

A model for the production of sister-chromatid exchanges is presented, based on the idea that double-strand breaks are generated at junctions between a completely duplicated replicon cluster and a partially duplicated replicon cluster. Agents that induce absolute blocks to DNA fork displacement will cause this condition to persist longer than normal, whereas agents that inhibit initiation of whole clusters will rarely cause it at all. During the blunt-end repair of the double-strand breaks, sister-chromatid exchange would be initiated when daughter strands of a duplicated cluster recombine with the parental strands of the partially replicated cluster. When the latter finishes replication, sister-chromatid exchange would be completed.