Figure 4

EBNA1 bound to FR blocks the progression of replication forks in transfected cells. A) Plasmids containing the SV40 replication origin, and FR regions with ten or 20 EBNA1-binding sites were linearized, and co-transfected into 293/EBNA1 cells with large T-antigen expression plasmid. A schematic representation of bidirectional replication fork movement from the SV40 origin is indicated above and below the linear transfected DNA, with the position of FR and the SV40 origin indicated. The leading strands from the SV40 origin are indicated as long arrows, and Okazaki fragments as the short arrows. Dark lines indicate unimpeded fork progression, while light gray lines indicated segments where diminished DNA synthesis is predicted. The positions and identities of restriction enzyme recognition sites to liberate fragments "ONE" and "TWO" from replicated DNA are shown. (B) Hirt extraction was sued to recover DNAs from transfected 293/EBNA1 cells that were subsequently digested with Dpn I and the specified restriction endonucleases to release fragments ONE and TWO, which were separated by electrophoresis, and quantified by Southern blot. Two independent experiments are shown with the migration of fragments ONE and TWO, and the number of EBNA1-binding sites in FR indicated. The TWO:ONE ratio is also shown.