Extended Data Fig. 6: SAMHD1 promotes the resection of DNA DSB ends.

a, Analysis of DNA end resection at the level of individual DNA fibres with the SMART assay. Control (shScr) and SAMHD1-depleted (shSAM) HEK293T cells were grown for 24 h in the presence of BrdU to label genomic DNA and DSBs were induced with 5 μg ml⁻¹ bleocin for 1 h. Cells were then washed and collected at the indicated times. DNA fibres were spread on glass slides and BrdU was detected without DNA denaturation. Representative images (three independent experiments) of BrdU tracks (red) 1 h after bleocin removal are shown. Scale bar, 5 μm. b, Quantification of BrdU track lengths (n = 200) in shScr and shSAM HEK293T cells treated with bleocin as in a. Median track lengths are indicated in red. ****P < 0.0001, Mann–Whitney rank sum test. c, Schematic of the U2OS single-strand annealing cell assay for DNA DSB repair. These cells carry a reporter vector in which an I-SceI site has been incorporated into a GFP gene, such that single-strand annealing-mediated repair events result in GFP fluorescence. d, U2OS single-strand annealing cells were transfected with siRNAs against SAMHD1, CtIP, or both, or with a control scrambled siRNA (Scr). They were then transfected with a plasmid expressing HA-tagged I-SceI under the control of a CMV promoter. Percentages of GFP-positive cells were quantified by flow cytometry and were normalized to the control cells. Error bars denote s.d. of three independent experiments. e, Expression of SAMHD1, CtIP and HA-tagged I-SceI in the experiment shown in d were monitored by western blotting (n = 3). f, Xenopus sperm DNA was incubated in Xenopus egg extract in the presence of 0.05 U μl⁻¹ of EcoRI for the indicated times then the chromatin was purified and analysed by western blotting for the indicated proteins. A representative experiment is shown (n = 3).

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