Skip to main content
. 2021 Oct 1;35(19-20):1356–1367. doi: 10.1101/gad.348667.121

Figure 5.

Figure 5.

Polα/primase activity in the absence of 53BP1 does not limit EXO1-dependent hyperresection. (A) Heat maps of END-seq signal ±3 kb around the top 200 AsiSI breaks in Lig4−/− and Lig4−/−53bp1−/− pre-B cells after 18-h AsiSI cutting. (B) Genome browser snapshot of DNA strand-separated END-seq in Lig4−/− and Lig4−/−53bp1−/− cells at a single AsiSI break (chr12: 111,039,088–111,039,096). More reads distal to AsiSI sites in Lig4−/−53bp1−/− compared with Lig4−/− indicates increased numbers and track lengths of resected breaks, consistent with the expected hyperresection phenotype. (C) Box plots of maximum resection lengths per AsiSI site (left) and ratio of resected breaks versus total breaks (right) as detected by END-seq after AsiSI induction with either no additional treatment, 5 µM aphidicolin (APH), or 1 µM DNA Polα inhibitor (POLAi) concurrent treatment. (D) Box plots of RPA single-strand DNA sequencing (SSDS) intensity in Lig4−/−53bp1−/− cells after 18-h AsiSI cutting with either NT, 5 µM APH, or 1 µM POLAi concurrent treatment. (E) Genome browser snapshot of SAR-seq signal at an individual AsiSI DSB with END-seq resection as reference (chr5: 115,630,000–115-635,000). (F) Heat maps of SAR-seq signal ±3 kb around AsiSI sites in Lig4−/− and Lig4−/−53bp1−/− cells after 18-h break induction. (G) Pearson's correlation of normalized read intensity at each AsiSI break location between Lig4−/− and Lig4−/−53bp1−/− SAR-seq from F. (H) Box plots of SAR-seq intensity in Lig4−/−53bp1−/− cells after 18-h AsiSI cutting with either NT, 5 µM APH, or 1 µM POLAi concurrent treatment. (I,J) Eighteen-hour AsiSI END-seq on either Lig4−/− (I) or Lig4−/−53bp1−/− (J) cells concurrently treated with DNA2 inhibitor (DNA2i), EXO1 inhibitor (EXO1i or C73), both, or NT. Resection signal at an individual AsiSI DSB (left, chr9: 63,756,000–63,761,000) and total resection signal aggregated and plotted for all 200 DSBs (right, Y-axis magnified to better visualize resection) are shown. All panels (except I and J, in which two independent replicate experiments were performed) show data sets from one experiment. A–H were independently corroborated using a separate Lig4−/−53bp1−/− clone in Supplemental Figure S5.