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. 2014 Sep 5;33(21):2521–2533. doi: 10.15252/embj.201488663

Figure 3. G-quadruplex structures are efficiently replicated in Xenopus egg extracts.

Figure 3

  1. Schematic representation of the G4 plasmid replication assay in HSS. Replication is started from primer A located 760 nucleotides (nt) from the G-quadruplex. Stalling of replication at the G-quadruplex structure will result in the accumulation of a 760-nt product, while G4 bypass first generates a 3,000-nt product that over time is ligated and supercoiled.
  2. G4G3N and non-G4C3p plasmid templates were replicated in HSS. Samples collected at the indicated times were separated on agarose gels and visualized with SybrGold (top panels). Gels were subsequently dried and visualized by autoradiography (bottom panels). The bands corresponding to the ssDNA, fully replicated but still nicked, and supercoiled dsDNA are indicated.
  3. Non-G4C3p, G4G3N, G4G5N, G4G15, and G4G23 plasmids were replicated in HSS, samples were taken at the indicated times, separated on 6% urea-PAGE gels and visualized by autoradiography. Products stalled at the G4 sequence (‘stalled’), linear molecules resulting from denatured nicked products (‘linear’), and closed supercoiled products (‘supercoiled’) are indicated.
  4. Non-G4C3p, G4G3N, G4G5N, G4G15, and G4G23 plasmid templates were replicated in HSS in the presence of 5 μM of Phen-DC3. Samples collected at the indicated times were separated on urea-PAGE gels and visualized by autoradiography.