The topography of DNA replication origins in Eukarya: GGN clusters, landmark nucleosomes, CDC6 and G4 structures

Abstract

BackgroundWe recently identified tandem repeats of GGN triplets as a sequence motif strongly enriched in the DNA replication origins (ORIs) of the Arabidopsis thaliana genome, where it correlates with the ORI strength quantified through short nascent strands sequencing. ResultsHere we show that clusters of four or more GGN repeats separated by short tracts (<7 nucleotides) are present in more than 65% ORIs of six model eukaryotic organisms: Leishmania major, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens. The measured percentages vary with the experimental technique adopted for ORI determination. For all studied techniques and organisms, ORIs are significantly prone to be located within or near strong GGN clusters, although these are neither necessary nor sufficient for ORI activation. Interestingly, for same experimental technique, the association between ORIs and GGN clusters was stronger al later developmental stages for all organisms for which we could perform this comparison. In accordance with a biophysical model of nucleosome positioning, the GGN clusters strongly favor nucleosome occupancy. At the same time, both GGN clusters and ORIs occur frequently within 1kb from nucleosome-depleted regions (NDR). We propose a structural model based on chromatin secondary structure in which the NDR and the well-positioned nucleosome at the GGN are close in space, which may favor functional interactions. We hypothesize that the presence of GGN at ORIs arose in part for promoting the above nucleosome organization Moreover, at least in Arabidopsis, the replication protein CDC6 is very strongly associated with GGN clusters. NMR experiments showed that clusters of at least four GGN can form G-quadruplex (G4) in vitro. Our data support the view that GGN clusters are formed through the interplay of mutational processes (GC skew at ORIs plus triplet expansion), and that similar mutational processes, i.e. AT skew at transcription start sites (TSS) and ORIs, might facilitate the formation of the NDR, thus favoring the evolvability of chromatin. ConclusionsGGN clusters are easily evolvable sequence motifs enriched at ORIs of eukaryotic genomes. They favor G4 secondary structure and a nucleosome organization that may unify the apparent discrepancy between ORIs of higher eukaryotes and yeast.