Position effects influencing intrachromosomal repair of a double-strand break in budding yeast

<div><p>Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (<i>r</i> = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (<i>CEN2</i>, position 238 kb) to the spindle pole body. Sequences at the same distance on either side of <i>CEN2</i> are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate <i>CEN2</i> tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.</p></div

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

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

Correlation between cell viability and distance of a homologous <i>LEU2</i> donor from (A) the left telomere and (B) the centromere (<i>CEN2)</i>.

<p>Pearson’s correlation test was conducted for either side of <i>CEN2</i> (including <i>CEN2</i>) respectively. Donor sites 10–12 (729 kb, 742 kb and 768 kb) are excluded from the analysis because viability had reached a plateau. <i>r</i> = 0.93 for right side of <i>CEN2</i>, and <i>r</i> = 0.95 for left side of <i>CEN2</i>.</p

Viability assay to assess repair efficiency for 12 intrachromosomal loci.

<p>(A) The scheme of viability assay. The <i>leu2</i>::<i>HOcs</i> was inserted at 625 kb on Chr2. The DSB could be repaired by an ectopic <i>LEU2</i> donor inserted on the same chromosome. The locations for the 12 donors were shown along Chr2. (B and C) Correlation between cell viability (%, shown in blue) and total contact frequency using ±25 kb window size around Chr2-DSB and ±10 kb (B) or ±20 kb window size around donor (C). Only 11 loci were analyzed in (B) since no productive contact was detected between ±25 kb around DSB and ±10 kb around site 4. Error bars indicate one SD from three independent experiments.</p

Effect of <i>cen2</i>Δ::<i>GAL-CEN3</i> on viability.

<p>Inactivation of <i>CEN2</i> significantly increased viability of two donors located close to <i>CEN2</i>. Error bars indicate one SD from three independent experiments.</p

New insights into donor directionality of mating-type switching in <i>Schizosaccharomyces pombe</i>

<div><p>Mating-type switching in <i>Schizosaccharomyces pombe</i> entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, <i>mat2-P</i> or <i>mat3-M</i>, is chosen to convert the expressed <i>mat1</i> locus. By measuring the biases in <i>mat1</i> content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (<i>mat2-M mat3-P</i>) or when the <i>SRE2</i> and <i>SRE3</i> recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from <i>mat2</i> and the <i>SRE2</i> enhancer, towards <i>mat3</i> and the <i>SRE3</i> enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD⁺-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD⁺-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to <i>mat2-P</i> in M cells, perhaps by inhibiting the <i>SRE3</i> recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.</p></div

Protein interaction network and genetic interactions.

<p>(A) An interaction network of newly identified and previously known mating-type switching factors (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.t001" target="_blank">Table 1</a>) was obtained from the STRING database (v10.0). The proteins are represented by nodes. Red and blue nodes show factors detected in this screen. White and black nodes show known mating-type switching factors that were not detected in this screen, either because the gene deletions were not in the library, or due to the set thresholds or human error. The line thickness represents the strength of the association (confidence > 0.6). It has been suggested that the presence of the DSB at <i>mat1</i> is lethal in deletion mutants of <i>rad51</i>, <i>rad52 and rad54</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.ref036" target="_blank">36</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.ref038" target="_blank">38</a>]). (B, C) Genetic interactions between <i>swi6</i> and the identified Class Ib genes. <i>mat1</i> content was quantified for the indicated double mutants with the <i>h</i>^<i>90</i> (B) or <i>h</i>^<i>09</i> (C) mating-type region. The relative P band intensities (P/(P+M)) were calculated from gels shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s006" target="_blank">S6 Fig</a>. Red bars represent means ± SD.</p

Determination of donor-choice preferences in mutants with the <i>h</i>^<i>09</i> mating-type region.

<p>The donor loci in <i>h</i>^<i>09</i> strains are swapped from <i>mat2-P mat3-M</i> to <i>mat2-M mat3-P</i>. Changes in <i>mat1</i> content caused by the Class Ib mutations identified here were estimated by multiplex PCR in the <i>h</i>^<i>09</i> background, from the band intensities shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007424#pgen.1007424.s004" target="_blank">S4 Fig</a>. Red bars represent means ± SD. Clr4, Raf1 and Rik1 are components of the H3K9 methyltransferase complex CLRC. Sir2 is a deacetylase. Swd1, Swd2 and Spf1 are components of H3K4 methyltransferase complex, Set1/Compass, Clr1, Clr2 and Clr3 are components of the deacetylase complex SHREC. One-way ANOVA was used to compare means of each sample to cntl, ***p < 0.001; ****p > 0.005.</p