Insights into the mechanisms of sister chromatid exchange formation

The DNA lesions responsible for the formation of sister chromatid exchanges (SCEs) have been the object of research for a long time. SCEs can be visualized by growing cells for either two rounds of replication in the presence of 5-bromo-2'-deoxyuridine (BrdU) or for one round with BrdU and the next without. If BrdU is added after cells were treated with a DNA-damaging agent, the effect on SCEs can only be analyzed in the second post-treatment mitosis. If one wishes to analyze the first post-treatment mitosis, cells unifilarily labeled with BrdU must be treated. Due to the highly reactive bromine atom, BrdU interacts with such agents like ionizing and UV radiation enhancing the frequency of SCEs. However, its precise role in this process was difficult to assess for a long time, because no alternative technique existed that allowed differential staining of chromatids. We have recently developed a method to differentially label sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) circumventing the disadvantage of BrdU. This technique was applied to study the SCEs induced by ionizing and UV radiation as well as by mitomycin C, DNaseI and AluI. This article is a review of the results and conclusions of our previous studies.     

In vivo sister chromatid differentiation and baseline sister chromatid exchanges in a mouse ascites tumour model.

Induction of differentially stained sister chromatids at G2/M and determination of baseline sister chromatid exchanges (SCEs) in ascites form of mouse sarcoma 180 cell line have been done by in vivo incorporation of 5-bromodeoxyuridine (BrdU) for two consecutive DNA replication cycles. The baseline SCE frequency is 6.24 at log phase of tumour growth.     

A schedule to demonstrate radiation-induced sister chromatid exchanges in human lymphocytes

Summary The reciprocal interchange between the chromatids of a chromosome, termed sister chromatid exchange (SCE), is considered to be one of the most sensitive and accurate cytogenetic parameters and respond to toxic chemicals at very low doses. But the response of SCE to ionizing radiation is very poor. Human lymphocytes fail to give SCE response when irradiated at G₀. Probably the primary lesions induced at G₀ do not remain available long enough to find expression as SCEs. Based on this assumption a schedule was developed using caffeine to demonstrate radiation induced SCEs. Following this schedule a dose-dependent increase in the frequency of radiation induced SCEs has been observed.     

Frequency of sister chromatid exchanges in cigarette smokers

Summary The effect of cigarette smoking on the frequency of sister chromatid exchanges (SCEs) was investigated in a group of adult men. It was observed that there was a significant increase in the mean SCE frequency per cell in smokers. Both the duration of smoking and the number of cigarettes smoked per day appeared to influence SCE frequency.     

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.     

Demonstration of spontaneous sister chromatid exchanges in vivo

The frequency of sister chromatid exchanges (SCEs) was examined as a function of bromodeoxyuridine (BUdR) concentration in vivo. Oneyear-old Wistar rats were continuously infused with BUdR through the tail vein for 24 h, sacrificed, and mitotic preparations prepared from femur bone marrow. It was observed that from the minimum concentration of BUdR which permitted accurate scoring (1.9 μg/g wt/h) to a BUdR concentration of 7 μg/g wt/h, SCE frequency remained constant. Above 7 μg BUdR/g wt/h SCE frequency increased, saturating at higher BUdR concentrations. The stability of SCE frequency at low BUdR concentrations is interpreted to indicate the existence of spontaneous SCEs in vivo.     

Effects of temperature on sister chromatid exchanges

Summary The influence of temperature on sister chromatid exchanges was investigated, and the results are discussed in connection with factors possibly involved in temperature-induced SCE-formation.Whereas the SCE frequency increased with increasing growth temperature in a cell line of Xenopus laevis (EAX), which permits the examination of great temperature differences, a Chinese hamster cell line (V-79) revealed a U-shaped temperature-response curve. In addition, it was found that cold treatment at 4°C caused an induction of SCEs in the V-79 cell line.Different BrdU concentrations had no effect on the temperature-induced SCE frequencies and mitomycin C led to an induction of SCEs parallel to the base-line values at different temperatures.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

Relationship of spontaneous chromosome instability and sister chromatid exchanges in Fanconi's anemia

The frequency of spontaneous instability of lymphocyte chromosomes of the first 2 mitoses, the rate of sister chromatid exchanges (SCEs), and the proliferative kinetics of lymphocytes were studied in a 6-year-old girl with Fanconi's anemia (FA) and in 4 healthy donors. The frequencies of aberrant cells and the total number of chromosome breaks in the FA patient decreased with cell transition from the first to the second mitosis. The FA lymphocytes had a slower proliferative kinetics and the level of SCEs was higher as compared with control. The probability of chromatid deletions at the sites of SCEs localization and in the dark and light stained chromatids was unequal. 33.8% of chromatid breaks were associated with SCEs. The data point to the relationship between SCEs and spontaneous chromosome instability in AF cells.     

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

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.     

Analysis of visible light-induced sister chromatid exchanges in 5-bromodeoxyuridine-substituted chromosomes

SCEs were studied in the chromosomes of Allium cepa L. stained by the FPG technique at the second and third divisions after BrdU-substitution during only the first replication round or the three consecutive cycles, respectively. Cells were cultured in the dark and exposed to visible light at different moments throughout the three cycles. The results obtained show that visible light-illumination, which has no apparent effect upon native DNA, is able to increase the frequency of SCEs in BrdU-substituted chromosomes. The comparison of expected and observed figures clearly reveals that BrdU-substituted DNA is the target of visible light. Finally, the formation of visible light-induced SCEs in BrdU-substituted chromosomes appears to be an S-dependent process, even though a post-replicational mechanism closely associated with semi-conservative S phase replication might be responsible.Abbreviations SCE sister chromatid exchange - BrdU 5-bromodeoxyuridine - Thd thymidine - FdU 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa - UV ultraviolet - CHO Chinese hamster ovary     

Sister chromatid exchanges in human cells and Chinese hamster cells: Evidence that the rate of sister chromatid exchanges is a function of ploidy

The frequency of sister chromatid exchanges (SCE) in the chromosomes of the diploidy and polyploidy of Chinese hamster cells and human cells has been studied using BUdR-DAPI (bromodeoxyuridine, 4′-6-diamidino-2-phenylindol) fluorescence. The rate of SCEs per cell under constant control conditions is in proportion to the ploidy levels. In addition, the frequency of SCEs observed in a given human chromosome (nos. 1) is also directly proportional to the number of such chromosomes presented in the cells. The mean of SCEs in human chromosome numbers 1 is very similar (0.46–0.48) for diploid, triploid, and tetraploid cells. The results suggest that the rate of SCEs is a function of cellular ploidy levels.     

Suppression of the frequencies of sister chromatid exchanges in Bloom's syndrome fibroblasts by co-cultivation with Chinese hamster cells

Summary The effect of co-cultivation of Bloom's syndrome fibroblasts with Chinese hamster ovary cells (CHO) on the incidence of sister chromatid exchanges (SCEs) was studied. The results show that suppression of the frequency of SCEs in Bloom's syndrome cells occurs only if cell to cell contact is present with CHO cells, without any effect on the SCE frequency in the latter.It is suggested that possible genetic heterogeneity between different Bloom's syndrome patients can be studied using the method of co-cultivation.     

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.     

Further analysis of the replication bypass model for sister chromatid exchange

Summary The replication bypass model for sister chromatid exchange (SCE) proposed by Shafer is examined in detail. When applied through two cell cycles, the model predicts that potentially observable SCEs induced during one S phase will then be cancelled and rendered undetectable during the subsequent S phase. This aspect of replication bypass is inconsistent with the observation of twin SCEs in tetraploid cells. Furthermore, the model cannot account for the efficient induction of SCEs by non-cross-linking chemical agents.     

Increased sister chromatid exchange in the peripheral blood lymphocytes of young women who smoke cigarettes

Rates of sister chromatid exchange in dividing human peripheral blood lymphocytes were determined and compared between smoking and non smoking young women between the ages of 16 and 25. Chromosomes block-stained with Giemsa were also examined for chromosome aberrations. A striking difference in the frequency of sister chromatid exchange was found between young women who smoked and those who did not. Smokers scored a significantly higher, F(1) = 15.99, p = 0.0015, rate of sister chromatid exchange than non smokers. Smokers scored a higher mean of SCEs per cell (12.771, SD 3.53) than non smokers (9.712, SD 2.53). Smokers also scored a higher range of SCEs (4 to 28) as opposed to non smokers (4 to 17). No statistical difference was found between smokers and non smokers for the frequency of chromosome aberrations. The significantly higher frequency of exchange in young smoking women may indicate that initial damage to the DNA in many of these women has probably already occurred, thus causing an increased risk of developing cancer later in life.     

3-Aminobenzamide-induced sister chromatid exchanges are dependent on incorporated bromodeoxyuridine in DNA

Data are presented establishing a direct correlation between 3-aminobenzamide-induced sister chromatid exchange (SCE) frequency and the level of bromodeoxyuridine (BrdU) incorporated into DNA. In addition, it is shown that most of the SCEs are induced in the second cell cycle, in which BrdU-containing DNA is used as the template for replication.     

Preferential occurrence of sister chromatid exchanges at heterochromatin-euchromatin junctions in the wallaby and hamster chromosomes

Chromosomes of two mammalian species, the white-throated wallaby and the rat-like hamster, possessed large amounts of constitutive heterochromatin which is detectable as C bands. By making use of this character, the frequency of sister chromatid exchanges (SCEs) was determined for the C band and the euchromatic regions of the chromosome. In both species, the distribution of SCEs in the euchromatin of chromosomes was found to be proportional to its metaphase length, while the number of SCEs localized in the C band regions was clearly fewer than expected on the basis of the relative length of those regions at metaphase. Many SCEs were, however, detected at the junctions between the euchromatin and the C band heterochromatin. All of these findings were consistent with previous observations on the Indian muntjac and the kangaroo rat chromosomes.     

Saccharomyces cerevisiae rad51 mutants are defective in DNA damage-associated sister chromatid exchanges but exhibit increased rates of homology-directed translocations.

Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Delta3'::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Delta3'::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.