Volume 9, Issue 4, Pages 835-846 (April 2002) Wild-Type Levels of Spo11-Induced DSBs Are Required for Normal Single-Strand Resection during Meiosis  Matthew J Neale, Madhu Ramachandran, Edgar Trelles-Sticken, Harry Scherthan, Alastair S.H Goldman  Molecular Cell  Volume 9, Issue 4, Pages 835-846 (April 2002) DOI: 10.1016/S1097-2765(02)00498-7

Figure 1 Diagrams of the Parental Reporter Construct and Derived Products The thick black line represents plasmid DNA; the thin black line is chromosomal DNA. (A) Both donor (arg4-bgl) and recipient (arg4-vde) cassettes contained an arg4 locus inserted into the natural ura3::Ty locus of SK1, creating direct repeats of URA3 sequence. (B) The VDE-DSB can form only in the arg4-vde allele. (C–F) Different products of DSB repair at the recipient locus: (C) gene conversion to ARG4, (D) gene conversion to arg4-bgl, (E) repair using flanking repeated sequences leading to ura3::Ty deletion product, and (F) repair using flanking repeated sequences leading to URA3 deletion product. Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)

Figure 2 Frequencies of Arg+ Colonies in RTG Experiments and Rate of VDE-DSB Formation (A and B) In both (A) allelic and (B) ectopic recombination experiments, the spo11f mutation led to a significant reduction in gene conversion to Arg+. The effect of the ndt80 mutation is to equalize the viability of SPO11 and spo11f strains. (C and D) Southern blots using BglII and EcoRV double digest of genomic DNA from the SPO11 and spo11f strains. Diagram to the right of (C) indicates the origin of fragments: B, BglII; R, EcoRV; P, position of probe. The 2.8 kb band contains a parental arg4-vde DNA fragment. The 2.3 kb band contains DNA from the arg4-nsp,bgl natural locus on chromosome VIII used as a loading control. (E) Quantification of the DNA in the parental arg4-vde band normalized to the amount of DNA in the loading control and doubled because only one chromosome V homolog contributes to the parental arg4-vde DNA fragment but both chromosome VIII homologs contribute to the loading control. While (A and B) the gene conversion frequencies differ, (E) the rate of VDE-DSB formation, expressed in terms of loss of the parental arg4-vde parent, is the same in both WT and spo11f. Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)

Figure 3 Analysis of the Proportion of VDE-DSB DNA and Deletion Product Present throughout Meiosis in WT and Different Mutant Strains (A–F) Southern blots using SpeI-digested genomic DNA from WT and mutant strains. The diagram to the right of (A) indicates the origin of the fragments: S, SpeI; P, position of probe; P/GC, parent or gene conversion. The 11.5 kb band contains the parental DNA from recipient (arg4-vde) and allelic donor (arg4-bgl) plus gene conversion products to either arg4-bgl or ARG4. The 7.8 kb band contains VDE-DSB DNA. The 2.3 kb band contains the ura3::Ty deletion product. The URA3 deletion product forms a band of 16 kb, which is usually too weak to be visible at this contrast but is detectable using the quantification software. The VDE-DSB band appears smeared in WT and sae2 strains (A, D, and E) but discrete in spo11f and hop1 strains (B, C, and F). The ura3::Ty deletion product is not shown for (E and F) the ectopic experiment because it is indistinguishable from the native ura3::Ty locus, which has no insert on the homolog. (G) Profiles of signal intensity running through the VDE-DSB band for the 4 hr time point. The graphs have been normalized to maximum signal and stacked for visibility. The wide shoulders in WT and sae2 reflect smearing, whereas the discrete bands from spo11f and hop1 produce symmetrical profiles. (H–J) Quantification of the amounts of DNA in (H and I) the VDE-DSB and (J) the ura3::Ty deletion product. The values plotted represent the proportion of total DNA in the lane doubled because only half of the chromatids probed contain the arg4-vde allele. The rate of appearance of deletion product is the same in spo11f and hop1 after a 1 hr delay (assuming a straight line for spo11f from 3–8 hr, R2 = 0.96, gradient = 6.5, and for hop1 from 4–8 hr, R2 = 0.97, gradient = 6.3). Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)

Figure 4 Slot Blot Analyses of Single-Stranded Intermediates (A) Diagram showing that probe signal is expected in native DNA only after the strand complementary to the probe is uncovered by resection passing beyond. (B) Example slot blot from a WT time course. In lane D, denatured DNA was loaded at one-tenth the concentration of DNA in the native lane, N. (C and D) The amount of signal obtained from the native DNA was normalized to ten times the signal obtained from the denatured DNA to obtain proportion of maximum signal. For both probes, maximum signal is achieved 2 hr earlier in spo11f compared to WT. Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)

Figure 5 FISH Analysis of Chromosome Pairing and Synapsis Using Detergent-Spread Meiocyte Nuclei after 6 Hr Sporulation (A and B) Differentially colored cosmid FISH signals specific to two different chromosome pairs, one shown in gray (#XI) and the second in black (#III, arrows). The gray background is color-inverted DAPI staining. Homologous chromosomes were scored as (A) paired when only one enlarged signal was seen per probe and nucleus and (B) separated when two signals were present for each probe and nucleus. (C–F) Nuclei after (C and D) DAPI staining and (E and F) anti-Zip1p-IF staining. DAPI-stained chromatin appeared (C) sausage-like in the SPO11 ndt80 strain which displayed (E) long Zip1p axes. Sausage-like DAPI morphology was largely absent in (D) spo11f ndt80 nuclei where (F) Zip1p was found in numerous dots throughout the chromatin and in polycomplexes (black oblongs). (G and H) Frequencies of paired homologous chromosome regions in SPO11 and spo11f strains, both homozygous for ndt80. The key is for both graphs. Bars: 5 μm. Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)

Figure 6 Assay to Determine the Distance Distribution of Resection Downstream of the VDE-DSB (A) Sample Southern blot from denaturing gel of WT DNA digested with HaeII. Bands labeled are: V, VDE-DSB; N/S, Nonspecific signal; P, Parent; R1–R6, progressively larger resection bands derived from successive destruction of HaeII sites as the length of ssDNA increases. In the diagram, the position of the VDE-DSB site, V, and HaeII sites, H, are indicated. (B) Quantification of the signal in bands R1, R3, and R6 expressed as a proportion of total signal in bands R1–R6. The blocks indicate the average data from two independent time courses; the range of data is shown by error bars. Molecular Cell 2002 9, 835-846DOI: (10.1016/S1097-2765(02)00498-7)