Repair in Trans is kinetically slower than repair in Cis.

<p>(A) Viabilities of the indicated strains (nd indicates not done). Data represent mean ± S.D. (n ≥ 6). (B-D) Kinetics of DSB repair in (B) Cis and Trans configurations, (C) Cis and Reverse-Cis configurations, and (D) Trans and Reverse-Trans configurations as determined by a quantitative PCR assay using primers shown schematically in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g001" target="_blank">Fig 1</a> and listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.s004" target="_blank">S1 Table</a>. The repair of <i>LE</i> and <i>U2</i> ends, which occurs predominantly by synthesis-dependent strand annealing, is indicated in the figure as SDSA, while the repair of the <i>URA3</i> and <i>ura3</i> ends, which occurs by single-strand annealing, is indicated as SSA. The amount of PCR product obtained from a repaired colony was used to make the standard curve for quantification. For Cis and Trans, data represent mean of a total of 6 PCR reactions from two independent time courses ± S.D. For Reverse-Cis and Reverse-Trans, data represent mean of three independent time courses ± S.D.</p

The kinetics of <i>LEU2</i> repair are independent of the repair outcome of another break.

<p>(A) Schematic representation of a modified Cis strain, tNS2607, which carries an <i>LE-HOcs-U2</i> cassette at the <i>can1</i> locus on Chr V, a <i>LEU2</i> donor on Chr III and an unrepairable <i>HOcs-URA3</i> break (instead of a <i>ura3-HOcs-URA3</i> cassette) on Chr XI. (B) Schematic representation of a strain which carries a <i>LE-HOcs-U2</i> cassette at the <i>can1</i> locus on Chr V and a <i>LEU2</i> donor on Chr III (YSJ119 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.ref013" target="_blank">13</a>]). This strain harbors a single HO break. (C) Kinetics of <i>LEU2</i> repair in the indicated strains, as determined by a quantitative PCR assay using primers shown schematically (black arrows) in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g001" target="_blank">1(B)</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.g003" target="_blank">3(A) and 3(B)</a>. The amount of PCR product obtained from a repaired colony was used to make the standard curve for quantification. (D) Data from (C) plotted after normalizing the amount of PCR product obtained at 12h time point for each strain to 100%. For Cis and Single Break, data represent mean of a total of 6 PCR reactions from two independent time courses ± S.D. For Modified Cis, data represent mean of three independent time courses ± S.D. The Single Break data shown in (C) has been published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005976#pgen.1005976.ref013" target="_blank">13</a>].</p

Adjacent <i>PIR</i> sequences interfere with GC repair in Trans.

<p>(A) Viabilities of the indicated WT, <i>sgs1Δ</i> and <i>msh6Δ</i> 147- and 265- Cis and Trans strains (nd indicates not done). Data represent mean ± S.D. (n ≥ 5). (B) Schematic representation of Chr XI features surrounding the site of insertion of the <i>LE-HOcs-URA3</i> cassette in the 147-Trans strain. The <i>LE-HOcs-URA3</i> cassette was inserted at position 147142 on the left arm of Chr XI. Orange lines represent the <i>PIR</i> genes and the arrowheads indicate their relative orientations. The distance of <i>PIR3</i> gene from <i>PIR1</i> and <i>LE-HOcs-URA3</i> cassette is indicated. The corresponding Cis strain contains a <i>NAT</i>-marked 5’ truncated <i>ura3-HOcs-URA3</i> cassette at position 147172. (C) Rad51 ChIP signal at the <i>LEU2</i> donor on Chr III representing the kinetics of strand-invasion by <i>LE</i> and <i>U2</i> ends in 147- Cis and Trans strains. Primers 300 bp and 200 bp upstream of the <i>LEU2</i> donor and 150 bp and 25 bp downstream of the <i>LEU2</i> donor were used to study the kinetics of strand-invasion by the <i>LE</i> and <i>U2</i> ends, respectively. (D) Schematic representation of the YMV80 SSA strain harboring an <i>HOcs</i> within the <i>leu2</i> gene at its endogenous locus, and a homologous <i>U2</i> sequence at the <i>his4</i> locus ~25 kb distal to the <i>LE-HOcs-U2</i>. Dotted lines indicate the regions spanning the Ty2 retrotransposon element, Ty1 LTRs (long terminal repeats) and tRNA genes that have been deleted in the TyΔ and IRΔ strains, respectively. TyΔ results in a net deletion of 4.7 kb. Figure not drawn to scale.</p