Evidence for rolling circle replication of tandem genes in Drosophila

Abstract

Extrachromosomal circular DNA (eccDNA) is one characteristic of the plasticity of the eukaryotic genome. It is found in various organisms and contains sequences derived primarily from repetitive chromosomal DNA. Using 2D gel electrophoresis, we have previously detected eccDNA composed of chromosomal tandem repeats throughout the life cycle of Drosophila. Here, we report for the first time evidence suggesting the occurrence of rolling circle replication of eccDNA in Drosophila. We show, on 2D gels, specific structures that can be enriched by benzoylated naphthoylated DEAE-cellulose chromatography and were identified in other systems as rolling circle intermediates (RCIs). These RCIs are homologous to histone genes, Stellate and Suppressor of Stellate, which are all organized in the chromosomes as tandem repeats. RCIs are detected throughout the life cycle of Drosophila and in cultured fly cells. These structures are found regardless of the expression of the replicated gene or of its chromosomal copy number.

Figure 1

Migration pattern of eccDNA and RCIs of histone genes. (A) Schematic outline of the 2D gel electrophoretic patterns of genomic DNA generated by populations of linear and circular molecules (3). Each arc consists of molecules sharing the same structure, but differing in mass. This analysis identifies double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), relaxed (open) circular molecules, supercoiled molecules and mitochondrial DNA (mtDNA). (B-C) A 2D gel of wild-type (Canton-S) adult DNA was hybridized with a cloned Drosophila histone H3 gene probe. A short exposure (B) revealed distinct circular multimers of 5 kb (arrowheads) and a massive arc of double-stranded linear DNA. In longer exposure (C) specific arcs emerging from each spot were detected (arrow, ‘eyebrow’). Similar results were obtained with histone H4 as a probe (data not shown).

Figure 2

RCIs are enriched by BND-cellulose chromatography. Genomic DNA from Drosophila adults was cleaved with EcoRV and subjected to BND-cellulose chromatography. The BND-cellulose-bound DNA was eluted by caffeine (caffeine, A, D) and was analyzed on 2D gel in comparison to a non-enriched sample of the same DNA (input, B, C, E, F), and quantification was performed as described in Materials and Methods. Hybridization with 28S rDNA probe reveals Y-shaped chromosomal replication forks of a 3.8 kb EcoRV fragment (arrow). Enrichment of replication forks is observed in the caffeine fraction (A) in comparison with their level in a similar amount of the input DNA (B) or even after a longer exposure of the input (C). Similarly, hybridization with histone H3 probe clearly demonstrates the enrichment of the putative RCIs (arrowhead) in the caffeine fraction (D) compared with their level in the input DNA (E) even at a long exposure (F). The numbers in parentheses refer to the relative values of the linear DNA signals in each comparison of two samples hybridized with the same probe, where the signal of the caffeine eluted DNA is 1.

Figure 3

Histone genes RCIs exist throughout the life cycle of Drosophila. 2D gel blots of DNA samples from (A) early embryos, (B) 3rd instar larvae, (C) adult flies, and (D) Kc cells were probed with histone H3. Arrows indicate RCIs.

Figure 4

RCIs corresponding to Stellate genes can be enriched by BND-cellulose and they co-migrate with histone RCIs. (A-B), Genomic DNA cleaved with BamHI was analyzed before (B) and after (A) BND-cellulose chromatography and probed with Stellate. DNA from adult males (Canton-S, C) and females (FM7, D) was analyzed on 2D gel. The membrane from D was re-hybridized with histone probe (E), and the merge of D and E (F) demonstrates the co-migration of the histone RCIs emerging from the 5 kb monomer, with those emerging from one of the Stellate multimers. Arrows indicate RCIs and arrowheads indicate a circular monomer (histone) or multimer (Stellate).

Figure 5

RCIs of Suppressor of Stellate genes. DNA from adult males (Canton-S) (A) and XXY females (B) was analyzed on 2D gel and probed with Suppressor of Stellate. Arrows indicate RCIs.

Figure 6

The abo mutation does not affect RCIs corresponding to histone genes. DNA from mutant abo¹/abo² flies (A) and their heterozygous sibling flies, which are a mixture of abo¹/Cy and abo²/SM1 and are labeled abo/+ (B) was analyzed on 2D gel and probed with histone H3. Arrows indicate RCIs. The samples in both A and B contain similar levels of linear DNA as was quantified by PhosphorImager, yet no clear difference in the level of RCIs was observed.