G4 motifs affect origin positioning and efficiency in two vertebrate replicators

Abstract DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation. Synopsis image Higher eukaryotic replication initiation sites are not specified by consensus DNA sequences but correlate with G-rich motifs that may form G-quadruplex (G4) structures. Genetic manipulation of such motifs in two chicken cell replicator sequences now provides first direct evidence for the requirement of G4 motifs in vertebrate replication initiation. Genetically tractable chicken DT40 cell lines and short nascent strand origin activity assays were used to assess the role of G4 motifs at two model origins. Activity of one origin depends on a critical 17-bp G4 sequence motif cooperating with a 200-bp flanking module. Point mutations affecting the stability of the G4 structure formed by this motif in vitro impair origin function in vivo. The orientation of the G4 motif determines the position of the replication start site. The G4 role in origin activity control is independently validated by a novel assay, based on replication timing shifts induced by a strong origin flanked with timing control cis elements. At the second tested model origin, two G4 motifs cooperate to drive highly efficient initiation.