Extended Data Fig. 2: SAMHD1 localizes to replication foci and binds nascent DNA.
From: SAMHD1 acts at stalled replication forks to prevent interferon induction
a, EdU (red) and SAMHD1 (green) foci in HeLa cells expressing HA-tagged SAMHD1 (SAMHD1–HA) or GFP–HA. Immunofluorescence microscopy was performed as indicated in Fig. 2a. Scale bar, 5 μm. n = 2. b, HeLa cells were labelled with 10 μM EdU for 10 min then processed for iPOND analysis. Proteins associated with nascent DNA were analysed by mass spectrometry. The number of peptides from SAMHD1 and other factors found associated with nascent DNA is indicated. The table summarizes the data from two independent experiments. c, Measurement of intracellular dNTP pools in shScr and shSAM HEK293T cells (two independent experiments). d, Production of recombinant His–xSamhd1 (see Supplementary Information) and characterization of the antibody raised against this protein. e, xSamhd1 associates with nascent DNA. Xenopus sperm DNA was incubated in Xenopus egg extract for 45 min then nascent DNA was labelled for 10 min with 40 μM biotin-16-dUTP in the absence or presence of 5 μM aphidicolin (Aph). Nascent chromatin was isolated on streptavidin beads and analysed by western blotting for the proteins indicated (see Methods). A representative experiment is shown (n = 2). f, Samhd1 binds chromatin in response to DSBs and aphidicolin. Left, Xenopus egg extracts were treated with the indicated doses of aphidicolin (μM), topotecan (T, 100 μM) or EcoRI (U μl−1). Chromatin-bound proteins were then analysed by western blotting. Right, histograms show relative signal intensity of Samhd1 on chromatin. Data are mean and s.d. from four independent experiments.
