Single Sister Chromatid Exchanges Research Articles

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 μM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 μM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.

Do the frequencies of sister chromatid exchanges in endoreduplicated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplicated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.

Analysis of single and twin sister chromatid exchanges in endoreduplicated normal and bloom syndrome B-lymphoid cells

Single and twin sister chromatid exchanges (SCEs) were analysed in the colcemid-induced endoreduplicated normal and Bloom syndrome (BS) B-lymphoid cells with diplochromosomes. In normal cells, an equal number of SCEs occur in each of the two cell cycles; the ratio of single (= 5.51 SCEs/cell) to twins (= 2.64 SCEs/cell) was 2:1 on the endoreduplicated-cell basis, and it was 1:1 on the diploid-cell basis. In contrast, in 29 endomitoses from one BS B-lymphoid line, a manyfold increase of single SCEs was detected and 139.4 single SCEs on the average were counted, whereas twin SCEs were rare and only 4.9 twin SCEs were countable. In BS cells, the ratio of single (= 139.4 SCEs/cell) to twins (4.9 SCEs/cell) was 28:1 on the endoreduplicated-cell basis, and it was 14:1 on the diploid cell-basis; the rates of S1 and S2 exchanges were 4.9 and 69.7 SCEs/cell, respectively. The present study strongly indicates that most of BS SCEs occur during the second cell cycle when BrdU-containing DNA is used as template for replication and that BrdU enhances BS SCEs.

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells.

Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5',8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7--2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost negligible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.

Sister chromatid exchange induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen.

To see if DNA crosslinks are involved in the induction of sister chromatid exchange (SCE), Chinese hamster ovary cells were exposed to two bifunctional alkylating agents, mitomycin C and 8-methoxypsoralen, and their monofunctional derivatives, decarbamoyl mitomycin C and angelicin. The data indicate that monoadducts, rather than crosslinks, are responsible for SCE formation. Furthermore, all agents but angelicin produced short-lived lesions that led to SCEs in the first period of DNA replication after treatment (twin SCEs), but not in the second (single SCEs). In contrast, angelicin, like methyl methanesulfonate and N-acetoxyacetylaminofluorene, produced lesions that lasted more than one cycle, indicating that several different types of DNA lesions are capable of SCE induction.

Recombinational DNA repair and sister chromatid exchanges

We show that a recombinational repair mechanism for DNA lesions can be expected to produce exactly the types of exceptions to the usually observed semiconservative segregation of newly synthetized DNA that have been reported in the literature. This removes the obstacles their occurrence appearance to present to the interpretation that the eukaryote chromosome is mononeme, containing but a single DNA double helix prior to replication. We further note that such a recombinational repair system would generate single sister chromatid exchange (SCE) events but not twin SCE events. This, along with other factors, complicates the interpretation of single: twin ratios in terms of any particular model of eukaryote chromosome structure.

Recombinational DNA repair and sister chromatid exchanges

We show that a recombinational repair mechanism for DNA lesions can be expected to produce exactly the types of exceptions to the usually observed semiconservative segregation of newly synthetized DNA that have been reported in the literature. This removes the obstacles their occurrence appearance to present to the interpretation that the eukaryote chromosome is mononeme, containing but a single DNA double helix prior to replication. We further note that such a recombinational repair system would generate single sister chromatid exchange (SCE) events but not twin SCE events. This, along with other factors, complicates the interpretation of single: twin ratios in terms of any particular model of eukaryote chromosome structure.

Influence of false twins on the ratios of twin and single sister chromatid exchanges

The relative frequency of single and twin sister chromatid exchanges has been of considerable interest because from it inferences have been made about the structure of chromatid subunits. The results of different experiments, however, yield greatly different ratios. Although the relative frequencies of single and twin exchanges should be independent of the rate of exchange, the frequency with which two singles will occur by chance at approximately the same chromosomal position, and thus mimic a twin, will depend upon that rate. The rate of exchange has indeed varied from one experiment to another, and so the contribution of false twins might account for some or all of the observed variability. Furthermore, the frequency of such false twins may have a large effect upon the single to twin ratio since not only do false twins increase the observed number of twins, but also they reduce the observed number of singles, thus affecting both numerator and denominator of the ratio. A formula for estimating the frequency of false twins has been derived on the assumption that exchanges occur at random along the length of the chromosome. When this is used to correct the published data, it is found that most of the variability from experiment to experiment remains. Evidently, there is some other factor that influences the relative frequencies of single and twin sister chromatid exchanges. The corrected ratio of single to twin sister chromatid exchanges in endoreduplicated cells is about 4:1, a value that is closer to the ratio expected for random rejoining of subunits under certain assumptions than for “polarized” rejoining. The implication of the observed ratios for chromosome structure is thus obscure.