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Fig. 1. Creation of an ectopic DNA replication origin. a Isothermal differential spectra (IDS; left panel) and circular dichroism spectra (CD; right panel) of potential OGRE/G4 sequences fond in the vicinity of replication origins. All tested sequences form G4 structures, as indicated by the strong negative peak around 295 nm and the two positive peaks around 240 and 273 nm (for IDS), and the strong positive peak around 260 nm (CD). The sequences are provided in Supplementary Table 1. b Ori1 that contains an OGRE/G4 element 240 bp upstream of the DNA replication initiation site (IS) was inserted by Cas9-stimulated homologous recombination into an origin-free region on chromosome 11 in NIH 3T3 mouse cells. The insertion of the 1907 bp fragment (marked in violet) occurred thanks to the two 500 bp homology arms (orange and green) present on the insertion template. The position of the primers (P) (sequences in Supplementary Table 2) used for the analysis of origin activity is also shown. c Ori1 activity in parental (control in black) and recombinant NIH 3T3 cells (in grey). As expected, a two-fold increase in DNA replication activity was detected in recombinant cells compared with parental cells, whereas the external origin Ori2 exhibited the same replication activity in both cell lines. Note that SNS activity was also detected at the 5′ and 3′ junctions of the insertion site, but not in the corresponding control regions. The background control regions Bcgd1 and 2 are located in origin-free regions; results are the mean ± SD of 3 independent experiments; p values were obtained using the two-tailed Student’s t test; *p ≤ 0.05, p > 0.05. d Analysis of DNA replication, using the DpnI digestion method and colony counting (“Methods” section), in an EBV episomal plasmid transfected in HEK293 cells that express EBNA1. DNA replication activity was assayed using EBV episomal plasmids that carry or not (Delta) the OriP origin, or a 500 bp fragment of Ori2 containing an OGRE/G4 element in the sense or antisense orientation. Results are the mean ± SD of 3–7 independent experiments; p values were obtained using the two-tailed Student’s t test; *p ≤ 0.05; ns not significant, p > 0.05 Image published in: Prorok P et al. (2019) © The Author(s) 2019. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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