Figure 2
From: MTBP phosphorylation controls DNA replication origin firing
Phosphorylation of MTBP at checkpoint kinase consensus sites inhibits genome replication. (A) Domain architecture of human MTBP with mutated consensus phosphorylation sites for ATR/M (S/T-Q, red) (amino acids T687, S761, S827, S858) and Chk1/2 (R/K-x-x-S/T, blue) (amino acids T531, T577, S579, T611, S738, S755, T781, T804, S808, S846). *, reported phosphorylations (phospho-site.org). Mutations to aspartate (D) or alanine (A) introduced in MTBP are indicated by colour-coded dots: MTBP-14A/D, 14 ATR/M and Chk1/2 mutated; 4/3/1A/D, all four, three or one ATR/M site(s) mutated; 10A/D, Chk1/2 sites mutated. (B) Chk1 in vitro phosphorylation of MTBP. Recombinant 6His-Treslin/TICRR-1-1258-MTBP-Strep was incubated with Chk1 in the presence of γ‐32P‐ATP and DMSO or Chk1 inhibitor AZD7762 and detected by autoradiography. Coomassie staining controlled loading (Load.); Rec., recombinant. (C) Flow cytometry density plots of HeLa Flip-In T-Rex cells expressing siMTBP-resistant C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-14D, MTBP-4D or no transgene, treated with control siRNA (siCtr) or siRNA against MTBP (siMTBP) and doxycycline, stained with anti-5 bromo-2′deoxyuridine (BrdU) after pulse-labelling and with propidium iodide (PI). Log./lin., logarithmic/linear scale; [AU], arbitrary units. MTBP-14D; MTBP-4D mutants, see A) (D,E) Quantification of replication activity (D) or cell cycle distribution (E) using BrdU-PI-flow cytometry as described in C. Error bars, SEM. n = 11 (no transgene); 9 (MTBP-WT); 8 (MTBP-14D); 3 (MTBP-14A); 8 (MTBP-4D); 5 (MTBP-4A); 3 (MTBP-10D); 4 (MTBP-3D); 3 (MTBP-1D); MTBP mutants: see A; significance tests: parametric, unpaired, two tailed student t-test. Significance tests in E) indicate differences in G2/M population distribution. (F) Whole cell lysates of stable cell lines described in C were analysed by immunoblotting using anti-MTBP (12H7), and Ponceau (Pon.) staining.
