The relation between chemically induced sister-chromatid exchanges and chromatid breakage.

Studies of classical chromosome aberrations and sister-chromatid exchanges (SCES) suggest independent mechanisms for the two events despite some common features. Examination of chromosome breakage caused by X-rays, visible light, and viruses has shown that few chromatid breaks are accompanied by SCEs at the sites of breaks. No similar observations were available for chemically induced breaks, but it has been reported that rat chromosomes exposed to dimethylbenzanthracene (DMBA) contained a preponderance of both aberrations and SCEs in certain specific regions, implicating a common process in their formation. These conclusions were drawn from a comparison of breaks induced in vivo with SCEs induced in vitro. However, we used 7 chemical mutagens to induce both chromatid breaks and SCEs in "harlequin" chromosomes of cultured rat and Chinese hamster ovary (CHO) cells and found that 25% of the 914 breaks scored were associated with SCEs. The proportion of breaks accompanied by SCEs is related to the overall SCE frequency and falls into the range predicted on the basis that breaks and SCEs occur independently. The reported association between sites for SCEs and aberrations also reflects secondary factors, such as induction of SCEs and aberrations during DNA synthesis in late replicating regions of the chromosomes.     

Frequency of sister chromatid exchanges in a balanced reciprocal whole-arm translocation

Summary The frequency of sister chromatid exchanges (SCEs) in the centromere of chromosomes involved in a whole-arm translocation t(1;19) was evaluated in altogether 911 metaphases of translocation carriers (n=5) and of normal controls (n=6). Comparison of the two groups reveals no significant differences in the SCE rate (x ²=3.06, n _f =1). The question as to whether the possible increase of the SCE rate at the translocation point could be detected by light microscopy is discussed. Parameters included in the discussion are the ratio of the SCE frequency at the translocation point to the SCE frequency at any of the possible breakage points in the centromeric region and the number of possible breakage points in the centromeric region.     

Mechanisms for sister chromatid exchanges and their relation to the production of chromosomal aberrations

By taking advantage of the fact that fluorescent light (FL) induces strand breaks only in bromodeoxyuridine(BrdU)-substituted DNA, and that those breaks eventually lead to the formation of sister chromatid exchanges (SCEs), the response of SCEs to FL was studied carefully in Chinese hamster chromosomes in which, out of four DNA strands, BrdU-substitution had occurred either in one or three strands. The FL-induced SCE frequency did not differ greatly between these two types of chromosomes. However, when they were submitted to caffeine treatment, a drastic increase in the frequency was detected in the trifilarly-substituted chromosomes while a significant decrease occurred in the unifilarly-substituted chromosomes. Based on these results, a working hypothesis was developed that the SCE can arise by at least two different mechanisms, one operating at replicating points probably utilizing the machinery of DNA replication, and the other acting only in the post-replicational DNA portion, probably in a similar fashion as assumed in a general model of crossing over in the eukaryote. These dual mechanisms may account for the discrepancy encountered in the explanations of the induction of SCEs by various exogenous agents as well as spontaneous SCEs. The present study also showed that some, but clearly not all, of chromatid deletions are the outcome of the failure to complete SCEs arising through these mechanisms.     

Enhanced G2 chromatid radiosensitivity in dyskeratosis congenita fibroblasts.

Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value.     

Progress towards understanding the nature of chromatid breakage

The wide range of sensitivities of stimulated T-cells from different individuals to radiation-induced chromatid breakage indicates the involvement of several low penetrance genes that appear to link elevated chromatid breakage to cancer susceptibility. The mechanisms of chromatid breakage are not yet fully understood. However, evidence is accumulating that suggests chromatid breaks are not simply expanded DNA double-strand breaks (DSB). Three models of chromatid breakage are considered. The classical breakage-first and the Revell "exchange" models do not accord with current evidence. Therefore a derivative of Revell's model has been proposed whereby both spontaneous and radiation-induced chromatid breaks result from DSB signaling and rearrangement processes from within large looped chromatin domains. Examples of such rearrangements can be observed by harlequin staining whereby an exchange of strands occurs immediately adjacent to the break site. However, these interchromatid rearrangements comprise less than 20% of the total breaks. The rest are thought to result from intrachromatid rearrangements, including a very small proportion involving complete excision of a looped domain. Work is in progress with the aim of revealing these rearrangements, which may involve the formation of inversions adjacent to the break sites. It is postulated that the disappearance of chromatid breaks with time results from the completion of such rearrangements, rather than from the rejoining of DSB. Elevated frequencies of chromatid breaks occur in irradiated cells with defects in both nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways, however there is little evidence of a correlation between reduced DSB rejoining and disappearance of chromatid breaks. Moreover, at least one treatment which abrogates the disappearance of chromatid breaks with time leaves DSB rejoining unaffected. The I-SceI DSB system holds considerable promise for the elucidation of these mechanisms, although the break frequency is relatively low in the cell lines so far derived. Techniques to study and improve such systems are under way in different cell lines. Clearly, much remains to be done to clarify the mechanisms involved in chromatid breakage, but the experimental models are becoming available with which we can begin to answer some of the key questions.     

Evidence that sister chromatid exchanges and chromatid breaks are two independent events

The relative frequencies of sister chromatid exchanges (SCE) and chromatid breaks in BrdU (5-bromodeoxyuridine) — sensitive site (lq22 lq23) in Chinese hamster cells after BrdU incorporation were studied. The results show that chromatid breaks do not follow the exchange hypothesis and provide evidence that chromatid breaks and SCEs are two independent events despite some common features.     

Localisation of 7-12-Dimethylbenz(a)anthracene induced chromatid breaks and sister chromatid exchanges in chromosomes 1 and 2 of bone marrow cells of rat in vivo

The frequency of chromatid breaks associated with sister chromatid exchanges at the break point was determined in rat bone marrow cells treated in vivo with 7–12 DMBA, during the late S phase of the cell cycle. The chromosomal aberrations and SCEs were scored in the same cells. Under the experimental conditions employed, more than 40% of the chromatid breaks were found to be associated with an SCE, a frequency expected according to Revell's hypothesis for the formation of chromatid breaks.     

Sister chromatid exchanges in cultured peripheral lymphocytes from twins

Summary Sister chromatid exchange points (SCE points) on individual chromosomes were studied in cultured lymphocytes from 11 monozygotic (MZ) and nine dizygotic (DZ) same-sexed pairs by means of sequential Q-banding and BUdR-Giemsa techniques. No statistically significant variation between unrelated individuals with respect to SCE points on specific chromosomes was found. Intrapair differences in the number of SCE points on specific chromosomes were not significantly smaller between MZ twin partners as compared with DZ partners. The results suggest that genetic factors do not play any major role in the frequency and distribution of SCE in normal subjects.     

Sister chromatid exchange assessment by chromosome orientation-fluorescence in situ hybridization on the bovine sex chromosomes and autosomes 16 and 26

Mammalian genome replication and maintenance are intimately coupled with the mechanisms that ensure cohesion between the resultant sister chromatids and the repair of DNA breaks. Although a sister chromatid exchange (SCE) is an error-free swapping of precisely matched and identical DNA strands, repetitive elements adjacent to the break site can act as alternative template sites and an unequal sister chromatid exchange can result, leading to structural variations and copy number change. Here we test the vulnerability for SCEs of the repeat-rich bovine Y chromosome in comparison with X, 16 and 26 chromosomes, using chromosome orientation-fluorescence in situ hybridization. The mean SCE rate of the Y chromosome (0.065 ± 0.029) was similar to that of BTA16 and BTA26 (0.065, 0.055), but was only approximately half of that of the X chromosome (0.142). As the chromosomal length affects the number of SCE events, we adjusted the SCE rates of the Y, 16, and 26 chromosomes to the length of the largest chromosome X resulting in very similar adjusted SCE (SCE(adj)) rates in all categories. Our results - based on 3 independent bulls - show that, although the cattle Y chromosome is a chest full of repeated elements, their presence and the documented activity of repeats in SCE formation does not manifest in significantly higher SCE(adj) rates and suggest the importance of the structural organization of the Y chromosome and the role of alternative mitotic DNA repair mechanisms.     

Elevated rates of sister chromatid exchange at chromosome ends

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.     

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.     

Non-uniform distribution of sister chromatid exchanges in human lymphocytes

To test whether sister chromatid exchange (SCE) scores on human chromosomes have a uniform distribution, simulated SCE scores were generated and compared with observed scores using log-linear models. The analysis was performed at the level of the chromosome groups. Using this method we first tested whether the number of SCEs was distributed uniformly, i.e. proportional to the relative length of the chromosomes. Refinements of this hypothesis were made by considering a variable region around a first SCE to be inert for other SCEs and by making the occurrence of an SCE on a chromosome dependent on the occurrence of another SCE on the same chromosome. In further analyses it was tested whether the number of SCEs was proportional to the number of G bands on a chromosome, or to the DNA content of the chromosomes. None of the tested hypotheses fitted the observed data, establishing the non-uniform distribution of these events.     

Sister chromatid exchange in patients with viral disease

Sister chromatid exchange (SCE) frequencies were studied in differentially stained chromosomes from lymphocytes of 17 patients with viral disease. The mean SCE score for the patients was 8.7 +/- 2.9 standard deviations. SCE scores were significantly elevated in the patients compared with the controls (p less than 0.01); however, variability in SCE means was observed in the patients. SCE elevations were also present in long term cultured Epstein Barr virus positive human B lymphocytes.     

Quantitative analysis of sister chromatid exchanges in the cell]

Assuming a random nature of distribution of sister chromatid exchanges (SCE) in a karyotype, the formulae have been obtained allowing the calculation of the number of SCE that are overlooked because of a limited resolving power of the SCE detection method. The results obtained mean that the actual number of SCE is more than the observed one, the part of overlooked exchanges being increased with the heightening of the SCE level. Taking into account overlook exchanges, the formula has been obtained that makes possible the calculation of the expected number of SCE observed in any group of chromosomes. These results were applied in the analysis of the SCE distribution among chromosomes. A better conformity has been obtained between the expected results and the observed ones, than under the assumption that the observed SCE are distributed in proportion to the lengths of chromosomes. The obtained formulae are of use in interpreting the lack of the observed SCE in small chromosomes and the excess of them in large ones.     

Sister chromatid exchange in the centromere and centromeric area

Central and peripheral sister chromatid exchanges (SCE) were evaluated separately in human phytohemagglutinin (PHA)-stimulated lymphocytes after culture for 72 h in 5-bromodeoxyuridine (BrdU) containing medium. At the same time, the length of chromosome No. 1 was measured in 10 metaphases per case and the mean value taken as a representative parameter for the contraction of chromosomes. The statistical analysis of regression revealed a close relationship between the percentage of SCE observed in the centromere and the contraction state of chromosomes (P less than or equal to 0.01). A statistically significant increase of central exchanges was seen in more condensed chromosomes, due to the difficulty in differentiating clearly between centric and pericentric exchanges. Consequently, if exchanges in the centromere are omitted from evaluation, this would lead to spuriously low SCE rates in more contracted chromosomes. In order to exclude the variable factor of chromosome contraction in SCE studies, we highly recommend inclusion of counts of central exchanges. Results obtained on chromosomes with twisted chromatids, a situation which tends to stimulate SCE, should be omitted.     

Sister chromatid exchanges are preferentially induced at expressed and nonexpressed common fragile sites

Summary To investigate the relationship between common fragile sites and sister chromatid exchange (SCE), lymphocyte cultures were treated with aphidicolin and bromodeoxyuridine (BrdU) and analyzed using a sequential GSCE staining protocol. A total of 1163 SCEs were mapped to their corresponding G-band sites, which were assigned to one of the following four categories: fragile sites expressed; fragile sites nonexpressed; nonfragile sites with breaks; or nonfragile sites with no breaks. The designated common fragile sites were found to be preferred locations for SCE formation, not only when these sites were expressed as visible gaps or breaks, but even when they were nonexpressed in the cell. SCEs were also more likely to occur at nonfragile sites with breaks than at nonfragile with no break sites. Further, SCEs were found to be distributed nonrandomly across fragile sites and nonfragile sites, and among the fragile sites, the high frequency SCE sites were highly correlated with the high frequency breakage sites. These data support the hypothesis of common steps in the mechanism of aphidicolin-induced SCE formation and common fragile site expression.     

Chromatid lesions and chromatid core morphology

A silver-staining technique revealed the core morphology of metaphase chromosomes of irradiated CHO cells with chromatid lesions (breaks, gaps). These cells were photographed before and after silver staining. As a rule, the core was not continuous in chromatid gaps, suggesting that the chromatid is broken in many so-called gaps. Ten cytogeneticists who were asked to classify chromatid gaps and breaks from photographs of chromosome lesions before silver core staining agreed in only 19 of 53 cases.     

Increased sister chromatid exchange in bone marrow and blood cells from Bloom's syndrome.

Bone-marrow cells from a patient with Bloom's syndrome cultured for 48 h in the presence of BudR exhibited a striking increase in the number of sister chromatid exchanges (SCEs) in comparison to that in the marrow cells of a patient with treated polycythemia vera (PV). Thus, it appears that an increased incidence of SCE in Bloom's syndrome occurs in various differentiated types of cells, not just blood lymphocytes, and constitutes the syndrome's most characteristic cytogenetic feature. In contrast, the incidence of SCE was not increased in marrow cells and lymphocytes of the particular PV patient studied here, whose cells did exhibit increased numbers of chromatid and chromosome gaps and breaks, presumably as result of the patient's earlier treatment. An increased frequency of SCE was demonstrated in Bloom's syndrome lymphocytes using both a technique based on BudR incorporation and one based on labeling with tritated deoxycytidine. This observation constitutes evidence against the increase of SCE being due to an unusual reaction to BudR. By conventional cytogenetic techniques, chromosome instability, including chromatid and chromosome breaks, but no homologous chromatid interchanges were also recognized in Bloom's syndrome bone-marrow cells incubated in vitro (without BudR) for either 1.k or 16 h. This observation points to the existence of chromosome instability in vivo.     

The induction of chromatid deletions in accord with the breakage-and-reunion hypothesis.

In the present experiments it has been possible to study large numbers of X-ray induced chromatid deletions, or breads, in Chinese hamster chromosomes and to discern whether or not a sister chromatid exchange also occurs at the point of breadage. Chromatid deletions are only infrequently associated with a sister chromatid exchange. This is contrary to the expectations derived from the exchange hypothesis of Revell. Pn the basis of this hypothesis, in which chromatid deletions are considered to be incomplete exchanges that occur in the necks of little loops in the chromosomes, 40% of the chromatid breaks are expected to be associated with sister chromatid exchanges. The present data are in accord with the conclusions drawn from the earlier autoradiographic experiments of HEDDLE AND BODYCOTE, and show that chromatide breaks can be accounted for on the basis of the breakage-and reunion hypothesis, with the majority being simple breaks and some being incomplete exchanges between two such breaks.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.