Figure 3. Analysis of HPV18E8ˉ RIs linearized with four different restriction enzymes.

1n, monomeric (8-kbp) linear molecules; 2n, dimeric (16-kbp) linear molecules. (a) 2D N/N AGE analysis of episomal HPV18E8ˉ RIs extracted from U2OS cells 3 days post-transfection and linearized with BglI, Bpu1102I, XmaJI, and PsyI. Black arrowheads, putative almost fully replicated, late theta RIs; black arrows, theta RIs; white arrows, non-theta molecules. The direction of the gel electrophoresis in the first (1D) and second (2D) dimension is indicated in the top left corner of the panel. (b) 3D N/N/A AGE analysis of XmaJI- and Bpu1102I-digested HPV18E8ˉ episomes extracted from U2OS cells 5 days post-transfection. P, parental strands; N, nascent strands. The direction of the gel electrophoresis in the third dimension (3D) is indicated in the top left corner. (c) The position of the BglI, Bpu1102I, XmaJI and PsyI enzyme recognition sites in the HPV18 genome (ori marks the approximate position of the origin of bidirectional theta replication¹³) and a scheme depicting bidirectional theta RIs digested with BglI/Bpu1102I and XmaJI/PsyI. (d) Migration patterns of nascent and parental strands originating from different types of RIs and parental strands of non-replicating molecules (represented by hemicatenanes) during 3D N/N/A AGE analysis³⁹,⁴¹.