Extended Data Figure 2. Estimation of efficiencies of the FR/EBNA1 and Tus/6xTer replication fork barriers.

a, Tus/Ter-mediated replication stall structures responsible for spots B and C. The relative abundance of the single stall spot B and the double Y stall spot C can be used to calculate the efficiency of the FR/EBNA1 replication fork barrier. b, Phosphorimager analysis of twelve independent Southern blot experiments (method described in Fig. 1b, see Source Data 3). Note: some images shown in Source Data 3 were also used in other figures. Areas B, B′, C and C’ are the same shape and size within individual panels. Size and shape of each area varies between panels. B: stall spot B. B′: background gel signal of same area as B. C: stall spot C. C’: background gel signal of same area as C. Relative intensity of spot B/B+C estimates the stalling efficiency at FR/EBNA1 and is calculated as:

$$(\mathrm{B}-\mathrm{B}\prime )/(\mathrm{B}+\mathrm{C}-\mathrm{B}\prime -\mathrm{C}\prime )\times 100\%.$$

The stalling efficiency at FR/EBNA1 is therefore 70% ± 2.2% (s.e.m.).

Relative intensity of spot C is calculated as:

$$(\mathrm{C}-\mathrm{C}\prime )/(\mathrm{B}+\mathrm{C}-\mathrm{B}\prime -\mathrm{C}\prime )\times 100\%.$$

c, Structure of p6xTer-2Ori plasmid. Stalled replication intermediates depict different combinations of FR/EBNA1 block/bypass and Tus/6xTer block/bypass. Spots B and B2 are defined as in the diagram. Spots C and C2 result from FR/EBNA1 bypass. Spot C2 requires successful arrest at both of the 6xTer arrays. Spot C results from bypass of one of the two 6xTer arrays. d, One of three independent experiments performed with p6xTer-2Ori. Methods as in Fig. 1b. Note presence of four stall spots in p6xTer-2Ori replicating in presence of wtTus. Double Y stall spots C and C2 and background signal C′ were quantified. Note: shape and size of each area is identical within an individual experiment, but varies between experiments (see Source Data 3). By considering only double Y stall spots (i.e., in which FR/EBNA1 bypass has occurred), the relative abundance of the double Y stall spots C and C2 are used to estimate the efficiency of the Tus/6xTer replication fork barrier. Let a = probability of the 6xTer array blocking the fork and b = probability of 6xTer bypass. Then:

$$a+b=1.$$

The probability of the two 6xTer arrays blocking each fork on one p6xTer-2Ori plasmid (generating spot C2) is a².

The probability of one 6xTer array being blocked and the second array being bypassed (generating spot C) is 2ab.

Relative densitometry of spots C and C2 (each with subtraction of background C′—Source Data 3) shows that spot C contributes 49.6% and C2 contributes 50.4% (s.e.m. 5.6%). Therefore:

$$\begin{array}{l}0.496a^2=0.504\times 2\mathit{ab}\\ a=0.67\end{array}$$

Therefore, the estimated efficiency of the Tus/6xTer replication fork block within the replicating plasmid is 67%. Note: the efficiency of the Tus/6xTer replication fork block within the chromosome is unknown.