<p>(A) Schematic illustration of inverted <i>ura4</i> repeat double RTS1 (<i>RuiuR</i>) construct. <i>RTS1</i> is a polar replication fork barrier. The triangular indent indicates the surface that prevents fork progression. (B) Cartoon representation of the expected replication intermediates (RIs) at the <i>RuiuR</i> locus as analysed by two-dimensional gel electrophoresis (2DGE). Left panel: RIs expected when the <i>Ase</i>I fragment indicated is replicated passively (no fork arrest at the <i>RTS1</i> barrier). Right panel - RIs expected in <i>RuiuR</i> cells upon fork arrest. (C) Left panel: control cells with no pause, demonstrating the position of the Y-arc. Middle and right panels: The <i>rtf1</i> ORF was inserted at the <i>urg1</i> locus in rtf1Δ cells by Cre-mediated cassette exchange to create YSM098 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083800#pone-0083800-t001" target="_blank">Table 1</a>). The strain was grown in EMM+LA at 30°C (asynchronous culture) and Rtf1 protein induced by the addition of uracil at 0.25 mg/ml. Samples taken at time-points shown. Chromosomal DNA was digested by A<i>se</i>I, and RIs were analysed by 2DGE.</p
