Sister chromatid exchanges in Tradescantia paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia paludosa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Sister Chromatid Exchanges in Tradescantia Paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia patudesa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Main repair pathways of double-strand breaks in the genomic DNA and interactions between them

Double-strand DNA breaks (DSBs) resulting from metabolic cellular processes and external factors pose a serious threat to the stability of the genome, but the cells have molecular mechanisms for the efficient repair of this type of damage. In this review, we examine two main biochemical pathways of repairing the double-strand DNA breaks in eukaryotic cells—DNA strands nonhomologous end joining and homologous recombination between sister chromatids or chromatids of homologous chromosomes. Numerous data obtained recently for various eukaryotic cells suggest that there is a complex interplay between the main DSB repair pathways, which normally facilitates efficient repair and maintenance of the structural and functional integrity of the genome, but which, at the same time, under conditions of exposure to genotoxic factors may induce increased genomic instability.     

Replication bypass model of sister chromatid exchanges and implications for Bloom's Syndrome and Fanconi's anemia

Summary A model of the sister chromatid exchange (SCE) process is outlined as a replication mechanism to bypass DNA crosslinks. The model suggests that when normal bidirectional replication advances from both sides towards a crosslink along the two opposite parental strands, the complementary parental strand segments can be temporarily displaced at each contralateral 5 side from the crosslink. The free ends produced in this first step will be terminally aligned but will have opposite polarity. The second step of the bypass can, however, be completed by either of two rejoining processes—terminal ligation of the free ends via nascent Okazaki pieces or aberrant complementation by overlapping the free ends. This bypass mechanism (1) allows replication to continue past a crosslink leaving it intact but (2) results in the switching of parental strands and their attached incomplete nascent strands above and below the crosslink site producing an exchange between sister chromatids. This model is compatible with the findings of current SCE studies using the new BUDR/stain techniques as well as with previous autoradiographic studies. It also suggests that the chromatid breaks and deletions in Fanconi's Anemia represent a defect in step two of the replication bypass mechanism and that the high frequency of SCE's and quadriradials in Bloom's Syndrome represent the SCE overload effects of a defect in crosslink repair.     

Mechanism of meiotic recombination in eukaryotes—A theory

It is proposed that in meiotic chromosomes single strand breaks of DNA originate either in the delayed regions of replicons or as a result of the excision activity of DNA polymerase during zygotene DNA synthesis. Rejoining of the break points belonging to non-sister chromatids takes place by switching over of the polymerase from one strand of DNA to another non-sister strand of the same polarity and gives rise to recombination intermediates (half-chromatid chiasmata). Strand migration in a recombination intermediate or copying of the same parental strand twice during zygotene as a consequence of a delay in copying the homologous strand would lead to gene conversion. Nicking of the cross strands (parental strands) in any recombination intermediate and subsequent repair leads to recombination for flanking markers. A possible way in which three-strand double crossovers occur and the process of recombination are discussed.     

Sister chromatid exchanges induced by light flashes to 5-bromodeoxyuridine- and 5-iododeoxyuridine substituted Chinese hamster chromosomes

Incorporation of 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR) into the chromosomal DNA of Chinese hamster ovary (CHO) cells during two rounds of replication causes sister chromatids to be differentiated so that they can be discriminated from one another by staining and morphology. Chromatids that contain BUdR or IUdR in both DNA strands stain lighter and are less condensed than their sister chromatids with only unifilar substitution. The halogenated pyrimidine nucleosides also induce sister chromatid exchanges that can be detected without autoradiography. The frequency of these exchanges is markedly increased by exposing the cells to light flashes.     

Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes

Summary The staining properties of unifilarly bromodeoxyuridine (BrdU)-substituted chromatids were compared using fluorescent-plus-Giemsa (FPG) staining methods. It was found that the staining intensity of chromatids which had incorporated BrdU in the next to last S-phase is less than that of chromatids whose BrdU-containing strand came from the last cell cycle. Thus, FPG-staining is not a function of the number of BrdU-substituted DNA strands alone. These findings lead to the conclusion that the primary point of action of PFG staining leading to sister chromatid differentiation (SCD) are chromosomal proteins which have been altered in the replication of BrdU-substituted DNA and that the demonstration of the SCD and replication patterns with the same staining procedure is based on different mechanisms.     

A model for ATP hydrolysis-dependent binding of cohesin to DNA

BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands. The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown. RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions. Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure. We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains. A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA. In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring. DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit. We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring. CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring. After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion. In anaphase, Scc1 cleavage opens the ring to release sister chromatids.     

RNase-dependent discontinuities associated with the crossovers of spontaneously formed joint DNA molecules in Physarum polycephalum

Transient four stranded joint DNA molecules bridging sister chromatids constitute an intriguing feature of replicating genomes. Here, we studied their structure and frequency of formation in Physarum polycephalum. By “3D gels”, we evidenced that they are not made of four continuous DNA strands. Discontinuities, which do not interfere with the unique propensity of the joint DNA molecules to branch migrate in vitro, are linked to the crossover, enhanced by RNaseA, and affect at most half of the DNA strands. We propose a structural model of joint DNA molecules containing ribonucleotides inserted within one strand, a gapped strand, and two continuous DNA strands. We further show that spontaneous joint DNA molecules are short-lived and are as abundant as replication forks. Our results emphasize the highly frequent formation of joint DNA molecules involving newly replicated DNA in an untreated cell and uncover a transitory mechanism connecting the sister chromatids during S phase.     

Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.     

Sister chromatid cohesion is required for postreplicative double-strand break repair in Saccharomyces cerevisiae.

The repair of DNA double-strand breaks by recombination requires the presence of an undamaged copy that is used as a template during the repair process. Because cells acquire resistance to gamma irradiation during DNA replication and because sister chromatids are the preferred partner for double-strand break repair in mitotic diploid yeast cells, it has long been suspected that cohesion between sister chromatids might be crucial for efficient repair. This hypothesis is consistent with the sensitivity to gamma irradiation of mutants defective in the cohesin complex that holds sister chromatids together from DNA replication until the onset of anaphase (reviewed in) . It is also in accordance with the finding that surveillance mechanisms (checkpoints) that sense DNA damage arrest cell cycle progression in yeast by causing stabilization of the securin Pds1, thereby blocking sister chromatid separation. The hypersensitivity to irradiation of cohesin mutants could, however, be due to a more direct involvement of the cohesin complex in the process of DNA repair. We show here that passage through S phase in the presence of cohesin, and not cohesin per se, is essential for efficient double-strand break repair during G2 in yeast. Proteins needed to load cohesin onto chromosomes (Scc2) and to generate cohesion during S phase (Eco1) are also shown to be required for repair. Our results confirm what has long been suspected but never proven, that cohesion between sister chromatids is essential for efficient double-strand break repair in mitotic cells.     

Actinomycin D effects on mitosis and chromosomes: sticky chromatids and localized lesions

When Indian muntjac and Chinese hamster cells in culture were treated with Actinomycin D (1 g/ml) for 1–2 hours, the sister chromatids, especially the distal segments, appeared to have difficulty separating in anaphase. The separated proximal segments progressively became stretched. The nucleolus organizer regions seemed to be most susceptible to stretching, and breaks in these regions were frequently observed. Electron microscopic observations showed that the sticky chromatids (and less frequently sticky chromosomes) contain connecting submicroscopic chromosome strands. When the treated cells were allowed to grow in a drug-free medium for several days, a high frequency of endoreduplicated mitotic figures was found. Chromosome and chromatid breaks and other aberrations were common, mainly localized at G band negative areas particularly nucleolus organizer regions.     

DNA damage in the kidney tissue cells of the fish Rhamdia quelen after trophic contamination with aluminum sulfate

Even though aluminum is the third most common element present in the earth''s crust, information regarding its toxicity remains scarce. It is known that in certain cases, aluminum is neurotoxic, but its effect in other tissues is unknown. The aim of this work was to analyze the genotoxic potential of aluminum sulfate in kidney tissue of the fish Rhamdia quelen after trophic contamination for 60 days. Sixty four fish were subdivided into the following groups: negative control, 5 mg, 50 mg and 500 mg of aluminum sulfate per kg of fish. Samples of the posterior kidney were taken and prepared to obtain mitotic metaphase, as well as the comet assay. The three types of chromosomal abnormalities (CA) found were categorized as chromatid breaks, decondensation of telomeric region, and early separation of sister chromatids. The tests for CA showed that the 5 mg/kg and 50 mg/kg doses of aluminum sulfate had genotoxic potential. Under these treatments, early separation of the sister chromatids was observed more frequently and decondensation of the telomeric region tended to increase in frequency. We suggest that structural changes in the proteins involved in DNA compaction may have led to the decondensation of the telomeric region, making the DNA susceptible to breaks. Moreover, early separation of the sister chromatids may have occurred due to changes in the mobility of chromosomes or proteins that keep the sister chromatids together. The comet assay confirmed the genotoxicity of aluminum sulfate in the kidney tissue of Rhamdia quelen at the three doses of exposure.     

Induction of chromatid breaks by carbon K-shell ultrasoft X rays

Chromatid breaks have previously been shown to be induced in G2-phase cells after exposure to ionizing radiation (X and gamma rays) as a linear function of dose, consistent with a single-event mechanism. DNA double-strand breaks (DSBs) are thought to be the initiating lesion, and experiments with a genetically engineered cell line containing a single DSB site also indicate that a single DSB is sufficient to induce a chromatid break. Although the precise mechanism of conversion of an isolated DSB into a chromatid break is not yet understood, it is known that a proportion of chromatid breaks result from rearrangements between sister chromatids. Here we report further evidence for the single-event hypothesis for the formation of chromatid breaks. The evidence derives from experiments in which chromatid breaks have been induced by exposure of Chinese hamster cells to ultrasoft carbon K-shell X rays. Since the energy of carbon K-shell X rays is not sufficient for the secondary electrons to span more than one DNA double helix, we conclude that single traversals, and hence single (complex) DSBs, are responsible for the formation of chromatid breaks. We find that, as for 60Co gamma rays, around 10% of the carbon K-shell X-ray-induced chromatid breaks have associated color switches at breakpoints, indicating that they arise through sister chromatid rearrangements.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.     

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.     

Molecular mechanisms of stress-induced hereditary variability

The molecular mechanisms of generation of stress-induced genetic recombinations and point mutations are considered. Due to the oxidative, temperature, radiation and other forms of stress, intensive modification of DNA bases occurs. Excision of the modified bases (hypoxanthine, uracil, pyrimidine photoproducts, methylated purines) leads to the formation of single-stranded gaps in DNA. If one DNA strand is damaged, there is high probability of its primary structure being completely restored. When the rate of lesions increases, the DNA can be damaged in the gap-related opposite sites of both strands. It is shown that in this case, the excision repair leads to a burst of recombinations and point mutations which may be concerned with the mispairings, double-stranded breaks, induction of SOS-response. With the increase in the rate of lesions, the possibility of the damage in self-complementary DNA sequences is also enhanced. This leads to formation of hairpin structures in the single-stranded DNA stretches. It is demonstrated that in these cases the repair results in development of deletions, insertions and clusters of point mutations predetermined by the primary DNA structure. Independent means of stress-induced mutations' occurrence seem to be the transposable elements. The stress-induced outbreaks of recombinations provide conceivably new variants of genotypes to be selected for the adaptation to new extreme conditions.