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. 2002 Dec;3(12):1168-74.
doi: 10.1093/embo-reports/kvf240. Epub 2002 Nov 21.

Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis

Sarit Cohen  1 , Marcel Méchali
Affiliations

Formation of extrachromosomal circles from telomeric DNA in Xenopus laevis

Sarit Cohen et al. EMBO Rep. 2002 Dec.

Abstract

Instability and plasticity of telomeric DNA, which includes extrachromosomal DNA, are usually correlated with the absence of telomerase and with abnormal growth of mammalian cells. Here, we show the formation of extrachromosomal circular DNA of telomeric repeats (tel-eccDNA) during the development of Xenopus laevis. Tel-eccDNA is double-stranded relaxed circles composed of the vertebrate consensus telomeric repeats [TTAGGG](n). Its size varies from <2 to >20 kb and it comprises up to 10% of the total cellular telomere content of the early embryo (pre-MBT stage). The amount of tel-eccDNA is reduced in later developmental stages and in adult tissues. Using a cell-free system derived from Xenopus egg extracts, we show that tel-eccDNA can be formed de novo from the telomere chromosomal tracts of sperm nuclei and naked DNA in a replication-independent manner. These results reveal an unusual plasticity of telomeric DNA during normal development of Xenopus.

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Figures

Figure 1
Figure 1
Tel-eccDNA is detected in Xenopus early embryos. (A) 2D gel electrophoretic patterns of genomic DNA generated by populations of linear and circular molecules (Cohen and Lavi, 1996). Each arc consists of molecules sharing the same structure but differing in mass. dsDNA, doublestranded DNA; ssDNA, single-stranded DNA; mtDNA, mitochondrial DNA; open circles, circular double-stranded DNA; supercoiled, double-stranded supercoiled DNA. (B–D) DNA from Xenopus early blastula (2000-cell stage), mixed with pUC18-derived plasmid relaxed-circle size markers (see text) and separated on a 2D gel. The blot was hybridized with telomeric repeat probe (TTAGGG)5 (B) and then with the plasmid probe (pUC18), to identify relaxed circles (C). The plasmids range from 2.7 kb (open arrowhead) to 22.4 kb (solid arrowhead). The solid arrow points to the relaxed circles, and the dashed arrows point to the linear DNA. (D) Co-migration of the telomere-homologous DNA arc with the circular markers is demonstrated by the superposition of (B) and (C).
Figure 2
Figure 2
Tel-eccDNA is resistant to S1 nuclease. Total genomic DNA from Xenopus early embryos was mixed with 1 μg of φX174 single-stranded DNA and treated with S1 nuclease. A sample was taken before adding the enzyme (A and D), and the other samples were taken after 5 min (B and E) and 30 min (C and F) of incubation. DNA was analyzed on a 2D gel and hybridized with telomeric probe (A–C) and φX174 probe (D–E) to follow degradation of single-stranded DNA. The solid arrows point to the single-stranded circular form of φX174, and the dashed arrows point to its linear form.
Figure 3
Figure 3
Tel-eccDNA consists exclusively of telomeric repeats. Genomic DNA from Xenopus early embryos was digested with the frequent cutting enzymes MspI and CfoI (A and C) and analyzed by a 2D gel in comparison with uncut DNA (B and D). The second dimension was carried out by running the two lanes corresponding to the first dimension on the top and bottom of the same agarose gel. Tel-eccDNA was not affected by the enzymatic cleavage (A and B), whereas satellite 1 (Lam and Carroll, 1983) was extensively cleaved as the whole bulk of genomic DNA. In the uncut DNA (D), a ladder of circular multimers of the satellite 1 unit (multimers of 741–750 bp) is detected. The arrow points to the low-molecular-weight linear fragments of satellite 1 following the MspI–CfoI cleavage.
Figure 4
Figure 4
The level of tel-eccDNA is decreased during development. Genomic DNA from Xenopus embryos and Xenopus erythrocytes was separated on a 2D gel: (A and B) 590 ng of early blastula embryos (pre-MBT, 1000-cell stage); (C and D) 1.4 μg of late neurula (90000 cells per embryo); (E and F) 1 μg of erythrocytes DNA. The quantity of embryonic DNA was estimated from the embryonic stage and checked by hybridization with a genomic DNA probe in comparison with DNA calibrated at 260 nm and loaded on the same gel (data not shown). The blots were hybridized with telomeric (A, C and E) and satellite 1 (B, D and F) probes.
Figure 5
Figure 5
In vitro formation of tel-eccDNA from sperm nuclei and from naked sperm DNA. Sperm nuclei (270 ng of DNA) (A–C) or 300 ng of purified naked sperm DNA (D–F) were incubated for 2 h in 100 μl of low-speed Xenopus egg extract in the presence (C and F) or absence (A, B, D and E) of 50 μCi of [α-32P]dCTP. Following incubation, the DNA was extracted and analyzed by a 2D gel as well as non-incubated naked sperm DNA (G and H). The gel was hybridized with telomeric probe (A, D and G) and with satellite 1 probe (B, E and H). The gel that contained the radiolabeled samples was exposed to autoradiography (C and F). The solid arrows point to arcs of relaxed circles. In (E), a series of supercoiled multimers is also identified. The dashed arrow points to single-stranded DNA in the preparation of naked sperm DNA, which was previously shown to be irrelevant to eccDNA formation, as its degradation by S1 nuclease does not alter the production of eccDNA (Cohen and Méchali, 2001). The white arrowheads point to mtDNA that is present in the extract and was also labeled.
Figure 6
Figure 6
Kinetics of tel-eccDNA formation in vitro. (A) Sperm nuclei were incubated in egg extracts in the presence (open circles) or absence (solid squares) of 100 μg/ml aphidicolin. DNA replication kinetics was followed during a 2 h period by incorporation of [α-32P]dCTP. Samples were taken at the indicated times for 2D-gel analysis followed by hybridization with telomere probe (B).
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