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

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

γ-H2AX Formation around <i>MAT</i> Spreads to the RE Region in <i>MAT</i>a Cells.

<p>(A) γ-H2AX formed around the <i>MAT</i> locus after DSB induction by HO. JKM139 (<i>MAT</i><b>a</b>) lacking <i>HML</i> and <i>HMR</i> was grown in galactose and subjected to ChIP analysis with anti-γ-H2AX antibody. DNA was extracted from immune-precipitates with protein G-agarose, and IP signals around the <i>MAT</i> locus were quantified via real-time PCR using five primer pairs (−10 kb, 10 kb, 20 kb, 30 kb and 40 kb from the HO cut site). IP signal at each locus was normalized to that of a control locus <i>CEN8</i>. Y axis represents IP signal as fold increase relative to the IP signal at the same locus before HO induction (time zero). Each data point is the average of two separate experiments, with error bars representing the range of IP values. (B) γ-H2AX appeared around the RE region in <i>MAT</i><b>a</b>, but not in <i>MAT</i>α cells. JKM139 (<i>MAT</i><b>a</b>) and JKM179 (<i>MAT</i>α) cultured in galactose for an hour were subjected to ChIP with anti-γ-H2AX antibody as described in panel A. To test γ-H2AX PCR signals around the RE, primers pairs at various distances from RE were used. Error bars represent the range of IP values from two independent experiments. (C) Kinetics of γ-H2AX formation around RE in JKM139 (<i>MAT</i><b>a</b>). All experimental procedures are same as described in Panel A for primer pairs amplifying regions around RE. Two independent experiments were performed and error bars represent the range of IP values. (D) The level of γ-H2AX signals around RE at 1 hr after HO induction was compared among the wild type (JKM139), <i>tel1Δ</i>, <i>mec1Δsml1Δ</i> or <i>mec1Δsml1Δtel1Δ</i> strains.</p

Measure Donor Preference via a PCR-Based Assay.

<p>(A) Mating-type switch at the <i>MAT</i> locus. When RE is active in <i>MAT</i><b>a</b> cells, donor preference (<i>HML</i> usage) is 85∼90%. In contrast, <i>HML</i> usage reduces to only 10∼15% when RE is deleted. Donor preference is calculated using a formula (<i>MATα</i>/(<i>MATα+MATα-</i>B)). (B) A PCR-based strategy for measuring donor preference. Diagrams are shown for <i>MAT</i>α and <i>MAT</i>α-B. After galactose induction, DSBs at <i>MAT</i> can be repaired using either donor of <i>HML</i>α and <i>HMR</i>α<i>-</i>B. A primer pair (Yalpha105F/MATdist-4R) can only amplify <i>MAT</i>α or <i>MAT</i>α-B, but not <i>HML</i>α, <i>HMR</i>α-B, <i>MAT</i><b>a</b> due to specificities of these two primers. (C) Measure donor preference via a PCR-based assay. Both <i>MAT</i>α and <i>MATα-</i>B are PCR-amplified using the primer pair (Yalpha105F/MATdist-4R). The purified PCR products are digested with <i>BamH</i>I and checked on ethidium bromide stained agarose gel. RE is present in XW652, but deleted in XW676.</p

The Presence of RE Promotes DSB–Mediated Interchromosomal Gene Conversion and Accelerates Rad51 Synapse Formation.

<p>(A) The presence of RE promotes the usage of its adjacent inter-chromosomal donor for DSB repair. An HO cut site was previously introduced to the <i>Kpn</i>I site of the <i>LEU2</i> to generate <i>leu2::HOcs </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002630#pgen.1002630-Paques1" target="_blank">[54]</a>. YCSL001 (as depicted) contains the <i>leu2::HOcs</i> at the <i>can1</i> locus on chromosome V. HO-induced DSBs can be repaired via gene conversion using one of two donors: <i>LEU2</i>, inserted ∼12 kb proximal to RE on chromosome III, and <i>leu2-K</i>, lacking a <i>Kpn</i>I site, integrated as part of a Yip5 plasmid at the <i>ura3-52</i> locus. Cells were plated on YP-galactose to induce DSBs. The repaired region of each survivor was amplified by PCR, followed by <i>Kpn</i>I digestion to determine which donor was used for repair. The bar represents the percentage of repair events using either donor. Light blue bars show the use of <i>leu2-K</i> while dark blue bars indicate the use of the ectopic <i>LEU2</i> on chromosome III. YCSL003 is same as YCSL001, except that RE is deleted. For YCSL001 (RE⁺), error bars are calculated from three experiments; for YCSL003 (REΔ), values are the same for two experiments (20 colonies per experiment). (B) The presence of RE accelerates Rad51 synapse formation. In YSJ119 (as depicted), the <i>LEU2</i> on chromosome III is the only homologous donor to repair the DSB on chromosome V. YCSL014 is same as YSJ119, except that RE is deleted. Both YSJ119 (RE⁺) and YCSL014 (REΔ) were grown in galactose and subjected to ChIP with anti-Rad51 antibody. IP signal was amplified using a primer pair (YCL049p1+Leu2-91082), indicated by a red solid line, which is located at the left boundary of <i>LEU2</i> on chromosome III. IP signal was normalized to that of a control locus <i>CEN8</i>. Y axis represents IP signal as fold increase relative to the IP signal at the same locus before HO induction (time zero). Error bars indicate the range of two experiments.</p

Roles of Histones and Kinases in Donor Preference and a Model for FHA-Directed Regulation.

<p>(A) The effect of H4 or H2A phosphorylation sites on donor preference. <i>HML</i> usage was not altered in strains only containing mutated <i>h4-S1A</i> or <i>hta1-S122A-T126A-S129A</i>. Donor preference was measured using a PCR-based assay (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002630#pgen-1002630-g001" target="_blank">Figure 1B</a>). (B) The effect of Mec1/Tel1 or casein kinase II on donor preference. In a triple mutant strain (YJL054), donor preference is not different from wild-type control (XW652). The <i>cka1::KAN</i>, <i>cka2::NAT</i>, pRS315-<i>cka2-8</i> (ts) are crossed into YJL019 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002630#pgen-1002630-g003" target="_blank">Figure 3A</a>) to generate the YJL119 strain. Both strains are first cultured at 25°C for overnight and then transferred to 37°C for 3 hour incubation. Galactose induction is performed for 1 hour and stopped by the addition of 2% dextrose. (C) A model for FHA-directed regulation of donor preference. After the generation of a DSB at <i>MAT</i><b>a</b>, we propose that a physical interaction between the FHA domain of Fkh1 and phosphothreonines of histones or bound proteins around the <i>MAT</i> will bring <i>HML</i> to the vicinity of the DSB, therefore allowing <i>HML</i> to serve as the favored template for DSB repair. The tethering of <i>HML</i> approximately 20 kb from the <i>MAT</i> can account for an almost 10-fold preference of <i>HML</i> usage over <i>HMR</i>.</p

The FHA Domain of Fkh1 Is Responsible for Donor Preference Regulation.

<p>(A) The strain construction strategy for YJL019, YJL020, YJL021 and YJL094. Fkh1 has two conserved domains: FHA and a forkhead DNA binding domain. We prepared three plasmid constructs by fusing LexA of pAT4 with different regions of Fkh1: pJL4 for LexA-FHA (aa 1–230 of Fkh1), pJL5 for LexA-interdomain (aa 163–302), and pJL6 for LexA-forkhead (aa 231–484), respectively. LexA-fused sequences from these plasmids were integrated to the <i>arg5,6</i> locus of ECY406 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002630#pgen-1002630-g002" target="_blank">Figure 2A</a>) to generate yeast strains YJL019, YJL020 and YJL021, respectively. For YJL094, LexA-fused sequences (LexA-FHA-R80A) from pJL8 were integrated. (B) FHA domain of Fkh1 is responsible for the regulation of donor preference. Donor preference was measured by Southern blot in panels B and C. (C) The phosphothreonine binding motif of FHA domain plays a critical role in the regulation of donor preference. XW652 and ECY406 serve as positive and negative controls, respectively.</p

A LexA System to Study Donor Preference.

<p>(A) Illustration of strain genotypes for ECY406, ECY457, ECY399 and YJL017. (B) LexA-Fkh1 regulates donor preference by binding to REΔ::LexA_BD4. ECY457 was constructed by integrating LexA-Fkh1 (from pEC16) to <i>arg5,6</i> of ECY406. Donor preference was measured by Southern blot using a Yα specific probe in panels B and C. XW652 serves as a wild-type control. (C) LexA-Fkh1 complements a <i>fkh1Δ</i> mutant (ECY399) to regulate donor preference presumably by binding to RE. The <i>arg5,6::LexA-Fkh1</i> was crossed into ECY399 to generate a strain YJL017.</p