(A) qPCR using primers spanning the HO cut site to monitor the efficiency DSB induction efficiency at 180 min after HO induction by uracil addition. (B) Diagram of the qPCR assay. Resection at the HO-induced DSB generates ssDNA through the ApoI site 168 nt from the HO cut cite, protecting from ApoI digestion. (C) Kinetics of resection assessed by qPCR and Rad52 loading. Percentage of chromosomes that have undergone 168 nt of resection at the HO cut cite over time, measured by the ApoI-site protection-based qPCR assay (bars, left y-axis). Error bars show SEM for three technical qPCR replicates. Corresponding data for Rad52-mCherry focus formation in the LacO resection assay (red line, right y-axis). (D–G) Representative images showing that LacO arrays as short as 1 kb in length are easily detectible under these imaging conditions. (D) is an image of LacO/LacI-GFP foci for a 10.3 kb LacO array (the same one used in the resection assay throughout). (E) is a comparision showing a still readily visible 1 kb LacO array, whereas (F) is a cell with LacI-GFP expression but no LacO array, showing the pan-nuclear background signal if LacI-GFP is allowed to diffuse throughout the nucleus without LacO binding. (D-E) are maximum intensity Z projections with similar exposure and contrast. (G) Is a control strain without any LacI-GFP expression that is imaged under longer exposure conditions and shown as a single Z slice in the middle of the cells to highlight the very faint autofluorescence in the cytoplasm but absent in the nucleus that is characteristic of fluorescent microscopy in S. pombe. Scale bars = 5 µm. (H) The rate of resection is equivalent on both the LacO and non-LacO sides of the HOcs in WT cells. Average long-range resection at the population level assessed by an orthogonal qPCR assay that quantifies the percentage of chromosomes that have undergone resection through ApoI sites at various distances from the HOcs. Resection to single-stranded DNA through an ApoI site protects that site from digestion by the double-strand-specific nuclease, ApoI. Error bars show 95% CIs for three technical qPCR replicates. (I) Resection rates, measured by a population-based qPCR assay, at various distances from the HOcs in exo1∆ and ctp1∆ cells 90 min after HO induction by uracil addition. Resection to single-stranded DNA through an ApoI site at the indicated positions relative to the HO cut site protects that site from digestion by the double-strand-specific nuclease, ApoI. Error bars show 95% CIs for three technical qPCR replicates. (J) On-target Rad52-mCherry foci form at reduced rates in exo1∆ cells. p-value shown is from pairwise two-tailed t-tests, using a Bonferroni correction for multiple comparisons. Number of biological replicates and counts of analyzed cells can be found in Supplementary file 2.