PGENETICS_ZMM_SGS1_MMS4_ReCombine_data

PGENETICS_ZMM_SGS1_MMS4_ReCombine_dat

Model of the relationship between Sgs1, Zip3 and Msh4 in CO-NCO fate.

<p>Models for how (A) WT, (B) <i>zip3</i> and (C) <i>sgs1</i> might produce the distribution of observed recombination types. Indicated in bold type are the main signatures observed. Arrow weight indicates the relative degree of partitioning through the pathway. Expected biased or unbiased cuts are indicated by asterisks or filled arrowheads.</p

Overview of meiotic recombination pathways.

<p>COs and NCOs normally form through two different pathways involving invasion of one of the resected 3′ ends of the DSB into a homolog followed by DNA repair (a) NCOs arise via SDSA where 3′ strand invasion is not accompanied by formation of a stable JM between the homologs. Disassembly of the SDSA intermediate allows reannealing and ligation to the other end of the DSB resulting in a NCO. (b) dHJ formation where the strand invasion is accompanied by stable JM formation. The majority of the dHJs are resolved into COs by biased cuts made to the two HJs followed by mismatch repair of the heteroduplexes. (Details about how heteroduplexes are resolved are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004690#pgen.1004690.s001" target="_blank">Figure S1</a>). Pairs of cuts are indicated by similar colored arrowheads. A small fraction of the dHJs can resolve to form NCOs by unbiased cutting of the two HJs. (c) In addition, a minority of events likely results from multistrand invasions. Event classifications (E#) are described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004690#pgen-1004690-g002" target="_blank">Figure 2</a>. Arrow weight indicates the degree of partitioning through the various pathways in WT.</p

Distribution of GCco and NCO tract lengths for <i>sgs1</i> and <i>zmm</i> mutants.

<p>(A) Comparison of GCco vs. NCO tract lengths for WT (B) Comparison of GCco vs. NCO tract lengths for <i>sgs1</i>. (C) NCO length distributions for <i>sgs1</i>, <i>msh4sgs1</i> and <i>zip3sgs1</i> (D) Box plot of GC_CO lengths. The dark line in each box represents median GC_CO length. Each box outlines lower and upper quartile. The whiskers denote the boundaries of the interquartile range and the circles outside the whiskers represent outliers (outside 1.5 times the interquartile range). The value inside each box is the median GC_CO length. (E) Comparison of GC_CO vs. NCO tract lengths for <i>msh4</i> (F) Comparison of GC_CO vs. NCO tract lengths for <i>zip3</i>.</p

Types of recombination signatures expected and observed by RecSeq.

<p>(A) The majority events are recombination signatures expected from normal SDSA and biased cleavage of JMs. Percentage of these events found for the various mutants are shown. Total events = E1–E7. (B) The minority events are recombination signatures that are found more rarely. Percentage of these events found for the various mutants are shown and can also be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004690#pgen.1004690.s006" target="_blank">Table S1</a>. WT has very few of these events. E1: Simple NCO is a 3∶1 tract on one chromatid and is not within 5 kb of another NCO or CO. E2: Simple CO is a CO with or without associated GC tracts and not within 5 kb of another CO or NCO. E3: Simple CO with discontinuous GC is a CO with or without associated GC tract with one or more GC within 5 kb and on one of the same chromatids as the CO chromatids. E4: Discontinuous NCO is two or more NCOs in a tandem fashion on one chromatid with regions of 2∶2 marker segregation separating them. E5 is an event where two or more COs or NCOs are within 5 kb of each other and involve only two chromatids. E5 is subdivided into E5A and E5B where E5A represents the signature of unbiased dHJ resolution and E5B represents all other E5 events. E6 is an event where two or more COs or NCOs are within 5 kb of each other and involve three chromatids. E7 is an event where two or more COs or NCOs are within 5 kb of each other and involve four chromatids. (Error bars – SE of proportions). T-test statistics comparing each mutant to another and WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004690#pgen.1004690.s007" target="_blank">Table S2</a>). (C) Average number of COs and NCOs per tetrad. Error bars, SD (D) Proportion of COs, NCOs and minority events out of total events.</p

<i>mms4-md</i> results in an increase in discontinuous CO and NCO events.

<p>(A) Histogram of interNCO distances showing an increased frequency of closely spaced NCOs (1^st bin) in <i>mms4-md</i> vs. WT. NCOs were identified with a 0 kb cutoff and then interNCO distances were calculated between each pair of adjacent NCOs. (B) Average number of simple NCO, discontinuous NCO, CO with GCco and CO with discGCco per tetrad plotted for WT and <i>mms4-md</i>. (Error bars – SD). (C) A random distribution of NCOs across the genome was simulated using the observed number of NCOs in WT and <i>mms4-md</i> tetrads respectively. CO positions were left unchanged. Distances were calculated between adjacent NCOs on same chromatid (NCO-NCO same), adjacent NCOs on different chromatids (NCO-NCO different), adjacent CO and NCO on same chromatid (CO-NCO same) and adjacent CO-NCO on different chromatids (CO-NCO different). Median distance for each category was plotted for WT and <i>mms4-md</i>. Asterisks indicate statistical significance (D) Percent of total NCOs and COs that show discontinuity in WT and <i>mms4-md</i>. (Error bars - SE of proportions).</p

Multiple strand invasions can account for discontinuities seen in <i>mms4-md</i>.

<p>(A) Multiple strand invasions or (B) a defect in mismatch repair could potentially account for appearance of discontinuities in <i>mms4-md</i>. (C) Depiction of the various components of a discontinuous NCO event. (D) Box plot for tract lengths of simple NCOs (E1). The dark line in each box represents median length. Each box outlines lower and upper quartile. The whiskers denote minimum and maximum data and the circles outside the whiskers represent outliers (outside 1.5 times the inter quartile range). Values for the median length are given. (E) Box plot of event length for the entire discontinuous NCO. (F) Box plot of individual tract lengths within the discontinuous NCO. (G) Box plot of gap lengths between discontinuous NCOs. (H) Representation of observed discontinuous NCO events in <i>mms4-md</i>, <i>msh2</i> and <i>mms4-md msh2</i>. Note that discontinuous events only make up 24% of all NCOs in a <i>mms4-md</i> mutant.</p

Simulation scripts

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

<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

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