MTBP phosphorylation controls DNA replication origin firing

Abstract

Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP's origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

Figure 1

Domain architecture of human MTBP with reported phosphorylation sites. Schematic of the MTBP protein. Reportedly phosphorylated consensus sites for ATR/M (S/T-Q, red, T687), Chk1/2 (R/K-x-x-S/T, blue, T577, S738, S755, T804, S846) and CDK (S/T-P, green, S539, T635, S639, S703, S707, T799) are indicated by vertical lines (see main text for references). S7M-N, -C, Sld7-MTBP amino and carboxy-terminal domains; blue oval, metazoa-specific domain; aa, amino acids; numbers, aa positions in human MTBP.

Figure 2

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.

Figure 3

MTBP inhibition by phosphorylation blocks origin firing, involving suppression of the S7M-C homo-dimerization domain. (A) Whole cell lysates and chromatin fractions isolated from Hela Flip-In T-Rex cells expressing siMTBP resistant C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-14D or no transgene after siMTBP or siCtr treatment were immunoblotted using antibodies against MTBP (12H7), Mcm2 and Cdc45. Coomassie staining controlled loading (Load.). (B) Whole cell lysates of the indicated siCtr or siMTBP-treated Hela-FlpIn cell lines were immunoblotted using anti-MTBP (12H7), anti-Chk1, anti-pS345-Chk1. Treatment with hydroxurea (HU) was used to control for ATR signalling. (C) Replication activity as measured by BrdU-PI flow cytometry of the indicated cell lines upon siMTBP treatment as described in Fig. 2D. n = 3, error bars: SEM, significance tests as in 2D/E. (D) C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-14D, MTBP-14A, MTBP-1A or MTBP-Cdk8bm were transiently transfected into 293T cells before analysis by anti-GFP immunoprecipitation (IP) and immunoblotting using antibodies against MTBP (12H7), Treslin/TICRR (117) and Cdk8; MTBP mutants: MTBP-1A, amino acid exchange T687A; MTBP-Cdk8bm, amino acid exchanges L620D, P622A, L623D, F632A, V633D, L634D, T635A. (E,F) Quantification of replication (E) or cell cycle distribution (F) in the indicated Hela Flip-In T-Rex cell lines as described in 2D/E. n = 4 (no transgene); 4 (MTBP-WT); 4 (MTBP-ΔC150); 4 (MTBP-5 m), 3 (MTBP-14D), 3 (MTBP-4D-GS4-GFP); 5 (MTBP-4D-GS4-GST); MTBP mutants: MTBP-ΔC150, C-terminal 150 amino acids deleted; MTBP-5 m, amino acid exchanges V306D, I309D, D313A, L314D, P315D.

Figure 4

Phosphorylation of MTBP at checkpoint kinase consensus sites inhibits origin firing in unperturbed cell growth conditions. (A) Flow cytometry PI-BrdU density plots of HeLa Flip-In T-Rex cells expressing siMTBP resistant C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT) or MTBP-14A treated with siMTBP, doxycycline, irradiated with 0 Gy or 20 Gy, and stained as in 2C. (i), density plots and (ii) quantification of replication signals, involving normalisation to cells irradiated with 0 Gy. Log., logarithmic scale; lin., linear scale; [AU], arbitrary units. Significance tests as in 2D/E. (B,C) Inter-origin distance measured by DNA combing of siMTBP-treated Hela Flip-In T-Rex cells expressing siMTBP resistant C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-14A or MTBP-cdk8bm. C(i), scatter plots of individual experiments; C(ii), quantifications of respective means; kbp, kilobasepairs. Error bars: SEM; n = 3; significance tests as in 2D/E. See also Supplementary Information Fig. S10.

Figure 5

MTBP is phosphorylated by cell cycle CDK and Cdk8/19-cyclin C. (A) Phosphorylation-mediated gel shifts of MTBP and Treslin/TICRR in mitotic cells depend on M-CDK. Cells arrested in mitosis with nocodazole (Noc) or asynchronous cells were treated for 30 min with DMSO, low (9 µM) or high concentrations (90 µM) of RO-3306 (RO.), roscovitine (Rosc.) or senexin A (Sen.). After cell lysis lysates were treated with lambda phosphatase (PPase) where indicated before immunoblotting using antibodies against MTBP (12H7) and Treslin/TICRR (148). (B) In vitro phosphorylation of MTBP by S-CDK. Recombinant 6His-Treslin/TICRR-aa1-1258-MTBP-Strep was incubated with Cdk2-cyclin A in the presence of γ‐³²P‐ATP and DMSO or CDK inhibitor roscovitine and detected by autoradiography. Coomassie staining controlled loading (Load.). Rec., recombinant. (C) S-CDK phosphorylation of MTBP depends on CDK consensus sites. C-terminally 3xFlag-TEV2-GFP-tagged MTBP-wild type (WT), MTBP-6A or control plasmid were transiently transfected into 293T cells before anti-GFP IP, incubation with buffer or Cdk2-Cyclin A in the presence of γ‐³²P‐ATP, and detection by autoradiography and immunoblotting using an antibody against MTBP (12H7). (i), autoradiogram; (ii), quantification of signal intensities of two independent replicates. WT signals were assigned the value 1 and control plasmid 0. MTBP-6A, amino acid exchanges S539A, T635A, S639A, S703A, S707A, T799A. (D,E) MTBP phosphorylation depends on binding to Cdk8/19-cyclin C. Native lysates of cells transfected as in C were immunoprecipitated and incubation with γ‐³²P‐ATP before detection by autoradiography or immunoblotting using an antibody against MTBP (12H7) and Cdk8. (F) Native lysates of cells transfected with the indicated MTBP versions and with Cdk8-cyclin C were analysis as in D in the presence of DMSO or Cdk8 inhibitor senexin A. (i), autoradiogram; (ii), quantification of signal intensities of three independent replicates. WT signal intensities were assigned the value 1 and Cdk8 inhibitor treatment 0.

Figure 6

Phosphorylation of MTBP on CDK consensus sites promotes origin firing in unperturbed growth conditions. (A,B) Quantification of replication activity (A) or cell cycle distribution (B) using BrdU-flow cytometry as described in 2D/E of indicated HeLa Flip-In T-Rex cell lines. Error bars, SEM. n = 3; MTBP mutants: MTBP-6A/D, see 5C. Significance tests as in 2D/E. (C) Whole cell lysates of stable cell lines described in A were analysed by immunoblotting using anti-MTBP (12H7), and Ponceau (Pon.) staining. (D) Inter-origin distance of cells as described in A measured by DNA combing as described in 4C. See also Supplementary Information Fig. S10.