Fig. 2: N70 undergoes ATR-dependent phosphorylation at the S279 cluster after exposure to various genotoxic insults.
From: A sophisticated mechanism governs Pol ζ activity in response to replication stress
a–d N70 was monitored by immunoblotting. In (b) cell extracts obtained 4 h after 10 J/m2 ultraviolet (UV) irradiation were incubated with CIAP at a concentration of 0, 62.5, 125, and 250 U/mL for 30 min at 37 °C prior to analyses. In (c) the gel mobility of N70 was monitored after 4 h indicated treatments. CPT, 3 µM camptothecin; HU, 2 mM hydroxyurea; MTX, 1 µM mitoxantrone; NCS, 3 µM neocarzinostatin; Etop, 50 µM etoposide. In (d) Prior to 10 J/m2 UV irradiation, cells were treated for 1 h with caffeine (Caf, 10 mM), 2-aminopurine (2-AP, 20 mM), VE821 (VE, 4 µM), KU-60019 (KU, 20 µM) and NU7026 (NU, 10 µM). Untreated cells were included in (c, d) as control (UC). e HCT116-3F-REV3L cells were transduced by the indicated shRNA virons for 3 days prior to analyses. f The indicated cells were grown in the medium with or without 1 µg/mL doxycycline (DOX) for 5 days prior to analyses. g HEK293 cells were transfected with various REV3L expression constructs. Fourty-eight hours after transfection, the UV-induced gel mobility shift of N70 was analyzed as described in (a). h A summary of the UV-induced phosphosites within N70 revealed by IP-MS analysis (top) and alignment of the sequences flanking the S279 cluster (bottom). Asterisks mark conserved residues. Serine at SQ motifs are highlighted in red. i REV3L protein was immunoprecipitated by anti-FLAG antibody from HCT116-3F-REV3L cells under denaturing condition following the indicated treatments. Phosphorylation of the S279 cluster was examined by western blot using a phosphopeptide-specific antibody. UC, untreated control; UV, 10 J/m2, 4 h recovery; HU, 2 mM hydroxyurea, 4 h exposure; DDP, 20 µM cisplatin, 4 h exposure. j ATR inhibitor (ATRi) pretreatment (12 h) suppresses the stress-induced phosphorylation of N70 at the S279 cluster. DM, 0.1% DMSO; ATRi, 4 μM VE-821. In (a–g, i, and j) each experiment was independently replicated at least twice, yielding similar results each time. Source data are provided as a Source data file.
