Analysis of replication fork blockage at RTS1 on a plasmid in cells deficient for SUMOylation, STUbL activity and Top1.
(A) Schematic of plasmid pREP3 containing RTS1, which is orientated so that it blocks the replication fork that approaches it from the replication origin (ars1) on its right as drawn . The bottom panel shows a replication fork originating from ars1 moving toward RTS1 and the position of the probe used for the analysis in C. (B) Schematic showing the main features of the 2D gel analysis of replication intermediates in C. These are: the arc of Y-shaped replication forks (Y); replication forks blocked/paused at RTS1 (P); replication termination where two opposing forks merge (T); and a spike of X-shaped DNA molecules that represent fully replicated conjoined DNAs. (C) 2D gel analysis of replication intermediates in the PstI-SacI fragment shown in A from wild-type or mutant cells as indicated. The strains are MCW1221, MCW4568, MCW5057, FO986, MCW6516, MCW5663, MCW4688 and MCW6514. (D) Amount of replication fork blockage/pausing as a percentage of the total Y-arc relative to wild-type. Values are the means of three independent experiments. Error bars represent standard deviations.