Data from: Reduced crossover interference and increased ZMM-independent recombination in the absence of Tel1/ATM

Meiotic recombination involves the repair of double-strand break (DSB) precursors as crossovers (COs) or noncrossovers (NCOs). The proper number and distribution of COs is critical for successful chromosome segregation and formation of viable gametes. In budding yeast the majority of COs occurs through a pathway dependent on the ZMM proteins (Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3), which form foci at CO-committed sites. Here we show that the DNA-damage-response kinase Tel1/ATM limits ZMM-independent recombination. By whole-genome mapping of recombination products, we find that lack of Tel1 results in higher recombination and reduced CO interference. Yet the number of Zip3 foci in tel1Δ cells is similar to wild type, and these foci show normal interference. Analysis of recombination in a tel1Δ zip3Δ double mutant indicates that COs are less dependent on Zip3 in the absence of Tel1. Together these results reveal that in the absence of Tel1, a significant proportion of COs occurs through a non-ZMM-dependent pathway, contributing to a CO landscape with poor interference. We also see a significant change in the distribution of all detectable recombination products in the absence of Tel1, Sgs1, Zip3, or Msh4, providing evidence for altered DSB distribution. These results support the previous finding that DSB interference depends on Tel1, and further suggest an additional level of DSB interference created through local repression of DSBs around CO-designated sites

Simulation scripts

Python scripts used to simulate crossover and DSB interference in Figure 6B

Absence of Tel1 alters the outcome of recombination.

<p>A) The average number of COs, NCOs, and all events (COs + NCOs) per tetrad is shown. COs include event types E2, E3, E5, E6, and E7 as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.ref053" target="_blank">53</a>] and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.g003" target="_blank">Fig 3</a>. NCOs include E1 and E4. B) The average ratio of COs to NCOs is shown for wt and <i>tel1Δ</i>. C) Histogram of distances between pairs of adjacent COs. D) Interference (1 –CoC) for COs in wild-type and <i>tel1Δ</i> tetrads. For each inter-interval distance, the CoC was calculated individually for all possible interval pairs genome-wide, and the average is plotted. For all plots, analysis of COs used data from 52 wild-type and 14 <i>tel1Δ</i> tetrads; analysis of NCOs and all events used data from 52 wild-type and eight <i>tel1Δ</i> tetrads. Error bars: standard error (SE).</p

<i>tel1Δ</i> and <i>sgs1Δ</i> show distinct recombination phenotypes.

<p>A) The average number of each product type is shown. Event types are as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.ref053" target="_blank">53</a>]. “Disc” = discontinuous. B) The average number of COs, NCOs, and all events is shown. COs include E2, E3, E5, E6, and E7. NCOs include E1 and E4. Plots of all contributing event types are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.s002" target="_blank">S2 Fig</a>. C) The average length of GC tracts at simple COs (E2) is shown. D) Histogram of the lengths of simple NCOs (E1). Error bars in all plots: SE. For all plots except analysis of COs in part B, data were derived from 52 wild-type, eight <i>tel1Δ</i>, nine <i>sgs1Δ</i>, seven <i>zip3Δ</i>, six <i>zip3Δ tel1Δ</i>, and six <i>zip3Δ sgs1Δ</i> tetrads. Analysis of CO frequency in part B used an additional set of six <i>tel1Δ</i>, four <i>sgs1Δ</i>, and 23 <i>zip3Δ</i> tetrads genotyped at lower resolution. Calculations of E8s in wild type used only the six wild type tetrads sequenced in our lab (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#sec017" target="_blank">Materials and Methods</a>). E) Sporulation frequency was measured in three independent cultures of each genotype, with the exception of <i>sgs1Δ</i> for which only two cultures were used. At least 300 cells per culture were counted. Average and SE are shown. The distribution of spores per ascus is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.s003" target="_blank">S3D Fig</a>. Viability was measured for at least 200 tetrads per genotype.</p

Reduced Crossover Interference and Increased ZMM-Independent Recombination in the Absence of Tel1/ATM

<div><p>Meiotic recombination involves the repair of double-strand break (DSB) precursors as crossovers (COs) or noncrossovers (NCOs). The proper number and distribution of COs is critical for successful chromosome segregation and formation of viable gametes. In budding yeast the majority of COs occurs through a pathway dependent on the ZMM proteins (Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3), which form foci at CO-committed sites. Here we show that the DNA-damage-response kinase Tel1/ATM limits ZMM-independent recombination. By whole-genome mapping of recombination products, we find that lack of Tel1 results in higher recombination and reduced CO interference. Yet the number of Zip3 foci in <i>tel1Δ</i> cells is similar to wild type, and these foci show normal interference. Analysis of recombination in a <i>tel1Δ zip3Δ</i> double mutant indicates that COs are less dependent on Zip3 in the absence of Tel1. Together these results reveal that in the absence of Tel1, a significant proportion of COs occurs through a non-ZMM-dependent pathway, contributing to a CO landscape with poor interference. We also see a significant change in the distribution of all detectable recombination products in the absence of Tel1, Sgs1, Zip3, or Msh4, providing evidence for altered DSB distribution. These results support the previous finding that DSB interference depends on Tel1, and further suggest an additional level of DSB interference created through local repression of DSBs around CO-designated sites.</p></div

PGENETICS_TEL1_ReCombine_data

Processed sequencing and microarray data from analysis of meiotic recombination in Saccharomyces cerevisiae. Genotypes analyzed are wild type, tel1, sgs1, zip3, msh4, tel1 zip3, and sgs1 zip3

Model for recombination pathway choice with and without Tel1.

<p>A) In contrast to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.g001" target="_blank">Fig 1</a> where DSB formation and CO designation were depicted as independent processes, we propose that formation of a SIC suppresses DSB formation nearby, so that later DSBs tend not to occur near a SIC. Early forming DSBs thus have a greater tendency to become interference-capable CO-designated sites and later DSBs tend to become NCOs or “non-interfering” COs. B) In <i>tel1Δ</i>, the number of DSBs is higher than in wild type and DSB distribution is less regular. A smaller fraction of DSBs becomes committed to the CO fate and marked by SICs; SICs still show an orderly distribution, as in wild type. DSBs not marked by SICs become NCOs or “non-interfering” COs, leading to decreased CO interference.</p

Overview of meiotic recombination.

<p>A) Major recombination pathways. A Spo11-induced DSB is resected to expose single-stranded tails. A 3’ tail invades a homologous duplex and is extended using the homolog as a template. Displacement of the invading strand leads to NCO formation by synthesis-dependent strand annealing (SDSA). Alternatively, capture of the second DSB end leads to formation of a dHJ. In wild type, dHJs are typically resolved as COs, but NCO formation is also possible. B) CO patterning. During or soon after DSB formation, a subset of DSBs becomes committed to the CO fate. These sites are marked by SICs and show interference. Subsequent steps convert CO-committed sites into COs. The majority of non-SIC-marked sites become NCOs, but some of them may also become COs.</p

Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I

ISSN:1534-5807ISSN:1878-155

The distribution of recombination events is altered in <i>tel1Δ</i>, <i>sgs1Δ</i>, and <i>zmmΔ</i>.

<p>A) Interference calculated as 1-CoC for a bin size and inter-interval distance of 25 kb is shown for COs only, NCOs only, or all events from whole-genome recombination data. <i>msh4Δ</i> data comprise seven tetrads sequenced in our lab and five tetrads genotyped by Mancera et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#pgen.1005478.ref051" target="_blank">51</a>]. B) Simulations were performed in which an interfering population of DSBs was first created, and then COs were selected from the DSBs. COs were selected either with or without additional interference. Remaining DSBs were considered NCOs. Failure to detect some events was simulated by removing 20% of all events and 30% of the remaining NCOs. Interference between all simulated DSBs or between “detectable” products is shown. Left: the strength of DSB interference was varied, and the strength of CO interference was selected to recapitulate observed interference between COs in wild type. Right: conditions were the same as on the left except no CO interference was incorporated. C) “Complex” events include the event types shown, and are events that could arise from more than one DSB. Randomized data consist of at least 10000 simulated tetrads per genotype in which the CO and GC tract positions in real tetrads were randomized. “With DSB landscape” indicates that event positions take into account DSB frequencies (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005478#sec017" target="_blank">Materials and Methods</a>). D) As in C, but includes only events involving four chromatids. Error bars: SE.</p