Extrachromosomal circles of satellite repeats and 5S ribosomal DNA in human cells

Abstract

Background: Extrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA.

Results: Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite.

Conclusions: These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity.

Figure 1

Alpha satellite sequences in extrachromosomal circular DNA from human cell lines. (A) A diagram of 2D gel electrophoretic patterns of genomic DNA generated by populations of linear and circular molecules heterogeneous in size. Each arc consists of molecules sharing the same structure, but differing in mass (Cohen and Lavi 1996). Hybridization with specific probes enables detection of specific sequences within the population of eccDNA. (B-E) Total DNA from human cell lines (indicated in each panel) was cleaved with EcoRI, digested with 'plasmid safe' DNase, analysed on two-dimensional (2-D) gels and hybridized with an 'all centromer' polymerase chain reaction product directed to the conserved region of the different types of alpha satellite. In all cases the probe hybridized with the linear DNA and with the arc that corresponds to open circles. (F-I) DNA from HEK-293 (human embryonic kidney cells) cells was mixed with a 10.8 kb plasmid prior to 2D gel analysis. Ethidium bromide (EtBr) staining of the gel reveals the three forms of the plasmid as well as the arc of linear DNA (I). These forms are visible upon hybridization with a plasmid probe (H). Hybridization with alpha satellite probe reveals the arcs of linear DNA and open circles (F). Note that the arc of open circles has a local deformation at the migration point of the over-loaded relaxed form of the plasmid. This is further confirmed by the merged image of panel F with a shorter exposure of panel H (G). White arrows indicate the plasmid forms; R = relaxed; SC = supercoiled.

Figure 2

Extrachromosomal circular multimers of the human SstI satellite DNA. Genomic DNA from HEK-293 (human embryonic kidney cells) (A) and LAN-1 (B) cells was cleaved with EcoRI, digested with 'plasmid safe' DNase, analysed on 2D gels and hybridized with SstI probe. A ladder of discrete spots indicates open circles in the size of multiples of the 2.5 kb repeating unit. Arrowheads indicate the multimers.

Figure 3

Absence of eccDNA homologous to the dispersed Alu repeats. HEK-293 (human embryonic kidney cells) DNA, was cleaved with EcoRI followed by 'plasmid safe' digestion and analysed on a two-dimensional gel. Hybridization of the blot with Alu probe revealed only the arc that corresponds to linear DNA (A). Rehybridization of the same blot with SstI probe revealed both linear DNA and eccDNA multimers (B). Arrowheads indicate the circular multimers.

Figure 4

Circular multimers of 5S rDNA and their rolling circle intermediates. Genomic DNA from HEK-293 (human embryonic kidney cells) digested with EcoRI and 'plasmid safe' DNase, was analysed on two-dimensional (2D)gel and hybridized with 5S rDNA probe. A series of spots forming a ladder indicate the presence of extrachromosomal circular multimers of the repeat (A). (B-D) For size analysis of the multimers, HEK-293 DNA digested with HindIII and 'plasmid safe', was mixed with plasmids of 2.7 kb, 7.76 kb and a 15.5 dimer of the latter prior to 2D-gel analysis. The blot was hybridized with 5S rDNA, revealing the circular multimers (B) and then rehybridized with a plasmid probe revealing the linear and the relaxed forms of the plasmids (D). (C) Merging the two autoradiograms verified that the eccDNA was indeed in the size of 5S rDNA multiples (see text). Arrowheads indicate multiples of 5S rDNA, and white arrows indicate plasmid markers. (E-F) Intermediates of rolling circle replication emerge from eccDNA. (E) Genomic DNA from LAN-1 cells, digested with EcoRI and 'plasmid safe', was analysed on 2D gel and hybridized with 5S rDNA probe. Long exposure reveals sigmoid arcs emerging from the 1n and 2n eccDNA (black arrows). Similar sigmoid arcs were found emerging from circular multimers of the 5 kb histone gene cluster of Drosophila (F), and were identified as rolling circle intermediates [17].