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PubMed1986

Concurrent unilateral chromatid damage and DNA strand breakage in response to 6-thioguanine treatment.

The delayed cytotoxicity of 6-thioguanine (TG) may relate to the arrest of cells in G2 upon completion of one cell cycle after drug exposure. In Chinese hamster ovary (CHO) cells, both the unilateral chromatid damage in G2 chromosomes, determined by induction of premature condensed chromosome condensation [Maybaum and Mandel, Cancer Res. 43, 3852 (1983)], and incorporation of TG into DNA resulting in DNA strand breakage [Christie et al., Cancer Res. 44, 3665 (1984)] were correlated with cytotoxicity. We have studied the correlation between strand breakage and unilateral chromatid damage in L1210 cells. DNA breaks were detected only when cells were treated with TG (0.25 microM) for one cell cycle time (12 hr) followed by 12 hr in drug-free medium containing [3H]thymidine (TdR) to label the DNA. After simultaneous incubation of cells with drug and label during the first or second 12-hr period, strand breaks were not found. Strand breaks increased with dose, which correlated with greater cytotoxicity (0.01 to 0.25 microM). Treatment of cells with 0.25 microM TG for 12 hr, and transfer to drug-free medium for 12 hr prior to making prematurely condensed chromosomes (PCC), resulted in unilateral chromatid damage. Prominent curving of G2 chromosomes with gapping and diffuse staining of one of the sister chromatids occurred. The 4-fold increase in the percentage of cells in G2 compared with control cells suggested G2 arrest. When cells were treated with TG for 12 hr and PCC made immediately, neither the arrest of cells in G2 nor unilateral chromatid damage was observed. These data suggest that strand breaks and unilateral chromatid damage occur in the second cell cycle after TG exposure and that this damage may be important in TG-delayed cytotoxicity.

Animals↗
PubMed2003

Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila.

BACKGROUND: Sister chromatid cohesion is needed for proper alignment and segregation of chromosomes during cell division. Chromatids are linked by the multiprotein cohesin complex, which binds to DNA during G(1) and then establishes cohesion during S phase DNA replication. However, many aspects of the mechanisms that establish and maintain cohesion during mitosis remain unclear. RESULTS: We found that mutations in two evolutionarily conserved Drosophila genes, san (separation anxiety) and deco (Drosophila eco1), disrupt centromeric sister chromatid cohesion very early in division. This failure of sister chromatid cohesion does not require separase and is correlated with a failure of the cohesin component Scc1 to accumulate in centromeric regions. It thus appears that these mutations interfere with the establishment of centromeric sister chromatid cohesion. Secondary consequences of these mutations include activation of the spindle checkpoint, causing metaphase delay or arrest. Some cells eventually escape the block but incur many errors in anaphase chromosome segregation. Both san and deco are predicted to encode acetyltransferases, which transfer acetyl groups either to internal lysine residues or to the N terminus of other proteins. The San protein is itself acetylated, and it associates with the Nat1 and Ard1 subunits of the NatA acetyltransferase. CONCLUSIONS: At least two diverse acetyltransferases play vital roles in regulating sister chromatid cohesion during Drosophila mitosis.

Acetyltransferases↗
PubMed2006

Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase.

BACKGROUND: The linkage between duplicated chromosomes (sister chromatids) is established during S phase by the action of cohesin, a multisubunit complex conserved from yeast to humans. Most cohesin dissociates from chromosome arms when the cell enters mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process is known as sister-chromatid resolution. Although two mitotic kinases have been implicated in this process, it remains unknown exactly how the cohesin-mediated linkage is destabilized at a mechanistic level. RESULTS: The wings apart-like (Wapl) protein was originally identified as a gene product that potentially regulates heterochromatin organization in Drosophila melanogaster. We show that the human ortholog of Wapl is a cohesin-binding protein that facilitates cohesin's timely release from chromosome arms during prophase. Depletion of Wapl from HeLa cells causes transient accumulation of prometaphase-like cells with chromosomes that display poorly resolved sister chromatids with a high level of cohesin. Reduction of cohesin relieves the Wapl-depletion phenotype, and depletion of Wapl rescues premature sister separation observed in Sgo1-depleted or Esco2-depleted cells. Conversely, overexpression of Wapl causes premature separation of sister chromatids. Wapl physically associates with cohesin in HeLa-cell nuclear extracts. Remarkably, in vitro reconstitution experiments demonstrate that Wapl forms a stoichiometric, ternary complex with two regulatory subunits of cohesin, implicating its noncatalytic function in inactivating cohesin's ability to interact with chromatin. CONCLUSIONS: Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.

Carrier Proteins↗
PubMed2005

Molecular analysis of sister chromatid recombination in mammalian cells.

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of double-strand breaks arising during replication and is thought to be important for the prevention of genomic instability and cancer. Analysis of sister chromatid recombination at a molecular level has been limited by the difficulty of selecting specifically for these events. To overcome this, we have developed a novel "nested intron" reporter that allows the positive selection in mammalian cells of "long tract" gene conversion events arising between sister chromatids. We show that these events arise spontaneously in cycling cells and are strongly induced by a site-specific double-strand break (DSB) caused by the restriction endonuclease, I-SceI. Notably, some I-SceI-induced sister chromatid recombination events entailed multiple rounds of gene amplification within the reporter, with the generation of a concatemer of amplified gene segments. Thus, there is an intimate relationship between sister chromatid recombination control and certain types of gene amplification. Dysregulated sister chromatid recombination may contribute to cancer progression, in part, by promoting gene amplification.

Animals↗
PubMed1998

Cohesion between sister chromatids must be established during DNA replication.

BACKGROUND: Cohesion between sister chromatids, which opposes the splitting force exerted by the mitotic spindle during metaphase, is essential for their segregation to opposite poles of the cell during anaphase. In Saccharomyces cerevisiae, cohesion depends on a set of chromosomal proteins called cohesins, which include structural maintenance of chromosomes 1p (Smc1p), Smc3p and sister chromatid cohesion 1p (Scc1p). Strains with mutations in the genes encoding these proteins separate sister chromatids prematurely and fail to align them in metaphase. This leads to missegregation of chromosomes in the following anaphase. RESULTS: In a normal cell cycle, Scc1p was synthesized and recruited to chromosomes at the onset of S phase. Using cells that expressed Scc1p exclusively from a galactose-inducible promoter, we showed that if Scc1p was synthesised only after completion of S phase, it still bound to chromosomes but failed to promote sister chromatid cohesion. CONCLUSIONS: Cohesion between sister chromatids must be established during DNA replication, possibly following the passage of a replication fork. Furthermore, Scc1p (and other cohesins) are needed both for maintaining cohesion during mitosis and for establishing it during S phase. Establishment of sister chromatid cohesion is therefore an essential but hitherto neglected aspect of S phase.

Cell Cycle Proteins↗
PubMed2004

The spindle pole body assembly component mps3p/nep98p functions in sister chromatid cohesion.

For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.

Acetyltransferases↗
PubMed2001

The XRCC2 human repair gene influences recombinational rearrangements leading to chromatid breaks.

PURPOSE: To test the possible involvement of the XRCC2 gene in the control of intra- versus interchromatid rearrangements leading to chromatid breaks in G2 cells by studying the colour-switch ratio (CSR) in harlequin-stained Chinese hamster irs1 cells. MATERIALS AND METHODS: The V79-4 mutant cell lines irs1 (XRCC2 mutation) and irs2 (XRCC8 mutation), two WT V79 lines and GT621-1 (irs1 transfected with the XRCC2 gene) were labelled with BrdU through two cell cycles, irradiated and sampled 1.5h after exposure. Metaphase spreads were analysed for chromatid break frequency and frequencies of colour-switch (colour-switch between chromatids at the break point) and non-colour-switch breaks, from which the CSR was calculated. RESULTS: Chromatid breaks were induced linearly with dose in all lines, and frequencies were elevated in irs1 and irs2 mutant cell lines when compared with WT lines. An XRCC2 transfected line (GT621-1) showed full radiosensitivity complementation with respect to frequencies of chromatid breaks. The CSR was significantly higher in irs1 (13.9%) than in the parental V79-4 (7.5%) or irs2 (4.9%) cells. GT621-1 cells showed partial, but significant complementation with respect to CSR (9.2%). CONCLUSIONS: It is concluded that the significantly higher CSR for the irs1 mutant than for the wild-type parental V79-4 line indicates the involvement of the XRCC2 gene product in the control of the rearrangement process leading to chromatid breaks.

Animals↗
PubMed2003

Comparison of bleomycin and radiation in the G2 assay of chromatid breaks.

PURPOSE: To compare bleomycin with radiation in the G2 chromatid break assay. Controversy exists in the literature about whether G2 bleomycin chromatid-break sensitivity links with cancer predisposition in the same way as the G2 chromatid radiosensitivity test (the so-called 'G2 assay'). Although bleomycin is referred to as a 'radiomimetic' agent, it differs from radiation in the way the damage is induced. MATERIALS AND METHODS: Epstein-Barr virus-immortalized lymphoblastoid cell lines from two head and neck squamous cell carcinoma patients, two breast cancer patients, two ataxia-telangiectasia patients and two normal control persons were used. Chromosomal damage was determined in cells exposed to 0.3-Gy radiation or 5 mU ml(-1) bleomycin. The numbers of chromatid breaks per cell and of aberrations per cell (i.e. breaks and gaps) were determined. RESULTS: A strong positive correlation was found between the two different damage inducers (r=0.99; p<0.001). This correlation was similar for both the breaks per cell and the total aberrations per cell. Inclusion of gaps in the scoring of chromatid breaks was associated with a higher variability of the data, but this did not influence the outcome of this study. CONCLUSIONS: Both bleomycin and radiation give the same sensitivity phenotypes as determined by the G2 assay of chromatid breaks. Thus, when no radiation facility is present, bleomycin seems to be a good alternative to radiation for this type of assay.

Bleomycin↗
PubMed2004

Sister chromatid exchanges in ring chromosomes following X-irradiation of human lymphocytes.

PURPOSE: To examine whether X-rays induce sister chromatid exchanges (SCE). MATERIALS AND METHODS: Peripheral lymphocytes irradiated in vitro or in vivo were cultured and treated with okadaic acid to generate premature chromosome condensation (PCC). When identical spreads were analysed using conventional Giemsa staining and pan-centromeric fluorescence in situ hybridization painting, ring chromosomes were observed. RESULTS: In PCC preparations, cells in the late G(2) phase and late M phase were observed. In late M phase cells, 17-20% of ring chromosomes lacked one chromatid (single-chromatid ring), irrespective of dose. Both the distribution patterns of centromeres in rings and intercentromere distances in dicentric rings indicate that a considerable number of single-chromatid rings might be formed by SCE occurring in a chromosome-type ring, thereby joining strands of two rings, followed by a transformation into one ring. These single-chromatid rings were less stable in vivo than chromosome-type rings. CONCLUSION: Single-chromatid rings visualized clearly using PCC techniques indicate SCE in the respective rings. Contrary to the conventional SCE-detecting technique, this approach does not require the use of bromodeoxyuridine, which itself leads to SCE. Some of the observed SCE might be secondary products resulting from the repair of radiation-induced DNA damage, while others may be spontaneous.

Cell Cycle↗
PubMed2000

Models for chromatid interference with applications to recombination data.

Genetic interference means that the occurrence of one crossover affects the occurrence and/or location of other crossovers in its neighborhood. Of the three components of genetic interference, two are well modeled: the distribution of the number and the locations of chiasmata. For the third component, chromatid interference, there exists only one model. Its application to real data has not yet been published. A further, new model for chromatid interference is presented here. In contrast to the existing model, it is assumed that chromatid interference acts only in the neighborhood of a chiasma. The appropriateness of this model is demonstrated by its application to three sets of recombination data. Both models for chromatid interference increased fit significantly compared to assuming no chromatid interference, at least for parts of the chromosomes. Interference does not necessarily act homogeneously. After extending both models to allow for heterogeneity of chromatid interference, a further improvement in fit was achieved.

Animals↗
PubMed1990

Similar kinetics of chromatid aberrations in X-irradiated xrs 5 and wild-type Chinese hamster ovary cells.

We have studied the kinetics of chromatid aberrations in cells of the Chinese hamster ovary (CHO-K1) derived, X-ray sensitive cell line xrs 5 irradiated in the G2 phase at 37 degrees C, as well as during a cell cycle extended by transient hypothermia at 33 degrees C. While a given X-ray dose was estimated to produce about 4 times as many chromatid break and twice the frequency of exchanges in xrs 5 cells as in the parent line, there was no difference between the lines in the rates of disappearance of chromatid breaks during G2 at either temperature; and similar patterns of chromatid exchange kinetics were observed in the two lines. Both the frequencies and distributions of chromatid breaks at different times after irradiation are consistent with the view that the disappearance of these during incubation represents a repair process. These results imply that the G2 chromosomal radiosensitivity of the xrs 5 mutant residues at the level of initial chromatid damage.

Animals↗
PubMedDate not supplied

Asymmetry in sister chromatids of human chromosomes.

Human metaphase chromosomes show identical lateral bands in sister chromatids when stained with quinacrine mustard or with Giemsa after trypsin treatment. A hybrid of these two methods produces banding patterns that are different in sister chromatids. Homologs may have similar patterns, so that a given chromatid is more like a homologous chromatid than its sister chromatid. It is suggested that asymmetry of sister chromatids may be important in regular mitotic disjunction.

Chromatids↗
PubMed1998

Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase.

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G1. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G1 results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G2 leads to cell death in subsequent metaphase-anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G1. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase.

Anaphase↗
PubMed2000

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange.

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

Animals↗
PubMed2005

Is mitotic chromatid segregation random?

The question of whether mitotic segregation of chromatids is random or programmed assumes great significance for cellular differentiation if one recognizes that sister chromatids may have epigenetic differences and carry them from one generation into the next. The literature was examined for evidence of non-random chromosomal and chromatid segregation. Many organisms were described as undergoing non-random homologue segregation in meiosis I. The explanations for these phenomena were attributed in some instances, to peculiarities of the meiotic spindle, though in some convincing experiments, the epigenetic heterochromatin of the kinetochores was implicated. The few existing descriptions of non-random mitotic segregation were also described. Existing literature on ultrastructural, immunohistochemical, and physiological features of the chromatid kinetochores during the mitotic process was searched for evidence of asymmetry or structural differences between sister chromatids, which is presented. Also reported are descriptions of how epigenetic changes and cell differentiation can influence centromeric function and ultimately, kinetochore function. Fundamental to the hypothesis of gene regulation presented here, is the assumption that genetic foci on different chromosomes interact, and must be proximate to each other and stereologically compatible for interactions to occur. Also described are spatial changes in chromosomal territories associated with function and differentiation. These territories can be in varying nuclear locations depending on gene function, and may show asymmetry between daughter cells. Despite evidence presented for the possibility of non-random chromatid segregation at mitosis, this question will remain unanswered until the matter is specifically addressed by experiment.

Animals↗
PubMed2004

Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion.

From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated.

Acetyltransferases↗
PubMed2001

Kinetics of chromatid break repair in G2-human fibroblasts exposed to low- and high-LET radiations.

The purpose of this study is to determine the kinetics of chromatid break rejoining following exposure to radiations of different quality. Exponentially growing human fibroblast cells AG1522 were irradiated with gamma-rays, energetic carbon (290 MeV/u), silicon (490 MeV/u) and iron (200 MeV/u, 600 MeV/u). Chromosomes were prematurely condensed using calyculin A. Prematurely condensed chromosomes were collected after several post-irradiation incubation times, ranging from 5 to 600 minutes, and the number of chromatid breaks and exchanges in G2 cells were scored. The relative biological effectiveness (RBE) for initial chromatid breaks per unit dose showed LET dependency having a peak at 55 keV/micrometers silicon (2.4) or 80 keV/micrometers carbon particles (2.4) and then decreased with increasing LET. The kinetics of chromatid break rejoining following low- or high-LET irradiation consisted of two exponential components. Chromatid breaks decreased rapidly after exposure, and then continued to decrease at a slower rate. The rejoining kinetics was similar for exposure to each type of radiation, although the rate of unrejoined breaks was higher for high-LET radiation. Chromatid exchanges were also formed quickly.

Cell Line↗
PubMed2003

[Genetic control of mitosis: features of origin of sister chromatid sets in anaphase in Drosophila melanogaster mutant line aar(V158)].

The effect of mutation aarV158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aarV158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.

Anaphase↗