Conditional replication fork-arrest assays.

<p>A. Diagrams of fork-arrest constructs. <i>Centromere</i>-proximal and <i>telomere</i>-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the <i>RTS1</i>-RFB. The <i>ura4⁺</i> gene is indicated in red and the arrow indicates its direction of transcription. Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: “t” and “ori” refer to the telomere and the replication origin 3006/7, respectively; “<“ and ”>” indicate the <i>RTS1</i>-barrier and its polarity (< blocks replication forks moving from the ori3006/7 towards the telomere, and > blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the A<i>se</i>I fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the <i>t>ura4-ori</i> construct does not result in a strong fork arrest as the <i>RTS1</i>-RFB is not orientated in the main direction of replication (see text for details).