Abstract
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called ‘finished’ genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.
Highlights
Centromere sequences evolve rapidly due to inevitable recombination processes undergone by tandemly repeated sequences but many centromere proteins are conserved[1]
Since a complete physical map across the centromere should extend from chromosome arm 3L to chromosome arm 3R, we set out to isolate bacterial artificial chromosome (BAC) clones that contain dodeca satellite, and to construct a comprehensive map around the dodeca satellite blocks using 20 restriction enzymes that do not occur within the dodeca satellite
Single and double genomic digests were size-fractionated by pulsed-field gel electrophoresis (PFGE) using a “Waltzer” apparatus[40], which gives sharp resolution up to 2 Mb
Summary
Drosophila centromere reveals the received: 23 January 2015 accepted: 21 July 2015. Published: 20 August 2015 prevalence of tandemly repeated sequences able to form i-motifs. We show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures. We determined that the G-rich dodeca satellite strand is able to fold into very stable intramolecular hairpin structures that are stabilized by the formation of noncanonical G:A pairs[28], and recently we have shown that the type B monomer of the human centromeric alpha-satellite[28] and the type A are able to form dimeric i-motif structures[29]. The distribution of restriction sites in the long-range map suggested that the region between the blocks consists of complex DNA sequences, while the flanking region of one of the blocks is likely to contain another putative block of repetitive DNA yet undescribed
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