Article Figures & Data
Figures
- Figure 1. UBA1 is recruited to an ATR-activating structure and is associated with ongoing replication forks.
(A) Experimental scheme: Biotinylated duplex DNA structures are incubated with nuclear extract in the presence of ATP. After 10 min at 37°C, DNA-bound proteins are pulled down using streptavidin-coated beads. To monitor protein ubiquitylation in vitro, reaction mixtures are supplemented with recombinant FLAG-ubiquitin. (B) Nuclear extracts were incubated with the indicated biotinylated DNA substrates. DNA-bound proteins were isolated, resolved by PAGE, stained with silver, and identified by mass spectrometry. The most abundant proteins are indicated. (C) Indicated proteins were pulled down with streptavidin-coated beads coupled to duplex DNA or gapped duplex DNA and detected by Western blotting. The duplex DNA and gapped duplex DNA substrates are represented schematically. Biotin (black circles) and streptavidin-coated beads (dented grey circles) are shown. (D) Indicated proteins were isolated by iPOND and detected by Western blotting. HEK293 cells were pulse-labelled with 5-ethynyl-2′-deoxyuridine (EdU) for 10 min and chased with thymidine for 60 min. In no click samples (no Clk), desthiobiotin-TEG azide was replaced by DMSO. (E) Bar plot showing average peptides intensities (MaxQuant label-free quantification) corresponding to the indicated proteins. HeLa S3 cells were pulse-labelled with EdU for 5 min and chased with thymidine for 120 min. Pulse experiment has been repeated three times and chase experiment two times. Error bars represent the standard variation. The UBA1 peptides identified are listed in Table S1.
- Figure S1.
UBA1 is recruited to DNA and promotes Chk1 phosphorylation. (A) Nuclear extracts prepared from HEK293 cells expressing UBA1-FLAG were incubated with the indicated biotinylated DNA substrates. DNA was pulled down using streptavidin-coated beads, resolved by SDS–PAGE, and probed for FLAG-UBA1 by Western blotting using anti-FLAG and anti-UBA1 antibodies, as indicated. (B) Nuclear extracts were incubated with recombinant FLAG-ubiquitin and the indicated biotinylated DNA substrates. DNA-bound ubiquitin conjugates were pulled down using streptavidin-coated beads and revealed by Western blotting using anti-FLAG antibody. (C) HeLa S3 cells were exposed to methyl methanesulfonate (MMS, 10 μg/ml) for 60 min in the presence of increasing concentration of PYR41 and probed for Chk1 and phospho-Chk1 (Ser345) by Western blotting. (D) Nuclear extracts were incubated with gapped DNA in the presence of [32P]-labelled NAD. Poly(ADP-ribosyl)ated proteins were resolved by PAGE and revealed by autoradiography. (E) Analysis of UBA1 and PCNA on nascent DNA. When indicated, HEK293 cells were pretreated with PARP1 inhibitors (10 μM of PJ34 or 10 μM of olaparib) and then pulse-labelled with EdU for 10 min. In no click samples (no Clk), desthiobiotin-TEG azide was replaced by DMSO. The fraction of UBA1 and PCNA associated with nascent DNA was retrieved using the iPOND procedure and revealed by Western blotting.
- Figure 2. UBA1 promotes Chk1 activation.
(A) DNA-bound proteins conjugated to FLAG-ubiquitin were isolated from nuclear extracts supplemented with solvent (DMSO) or PYR41 and immunoblotted with anti-FLAG antibody. The DNA substrates are represented schematically. Biotin (black circles) and streptavidin-coated beads (dented grey circles) are shown. (B) Immunoblotting of UBA1 and Chk1 in nuclear extracts prepared from HeLa S3 cells treated with siRNAs against UBA1 (siUBA1) or control (siCTR). (C) Ubiquitylated proteins bound to DNA were isolated from control and UBA1 knockdown cells and analysed as described in (A). (D) Western blot analysis of the indicated proteins isolated with DNA structures from protein extracts supplemented with PYR41 or solvent (DMSO). (E) DNA-bound proteins were pulled down from control or anti-UBA1 siRNA nuclear extracts and analysed by Western blotting as indicated. (F) HeLa S3 cells pretreated or not with increasing doses of PYR41 were exposed to 1 μM CPT for 120 min and the indicated proteins were probed by Western blotting. (G) HeLa S3 cells treated with siRNAs against UBA1 (siUBA1) or control (siCTR) cells were treated or not with 1 μM CPT for 120 min and probed for the indicated proteins by Western blotting.
- Figure 3. pADPr chains are required for UBA1 recruitment.
(A) Reaction mixtures were incubated with [32P]-labelled NAD, and DNA-bound proteins were pulled down and resolved by PAGE. Poly(ADP-ribosyl)ated proteins were revealed by autoradiography. (B) Autoradiography of proteins poly(ADP-ribosyl)ated in nuclear extracts and isolated with the indicated DNA substrates represented schematically. Biotin (black circles) and streptavidin-coated beads (dented grey circles) are shown. When indicated, reactions were performed in the presence of the PARP1 inhibitors PJ34 or olaparib. (C) Western blot analysis of the indicated proteins isolated with biotinylated DNA substrates from reaction mixtures supplemented, when indicated, with PJ34 or olaparib. (D) Nuclear extracts were pretreated with PARG when indicated and incubated with DNA substrates. The indicated proteins were isolated by DNA pull-down and revealed by Western blotting. (E) Western blot analysis of PARP1 and Chk1 in nuclear extracts prepared from cells treated with anti-luciferase (shCTR) or anti-PARP1 (shPARP1) shRNAs. (F) The indicated proteins were isolated along with biotinylated DNA substrates after incubation in protein extracts from cells treated with control (shCTR) or anti-PARP1 shRNAs, as indicated.
- Figure 4. Human UBA1 binds to pADPr chains.
(A) Homo sapiens UBA1, H2A, and BSA were spotted on a nitrocellulose membrane, incubated with [32P]-labelled pADPr, washed, and exposed to autoradiography. (B) Purified H2A, BSA, H. sapiens UBA1 (UBA1), S. cerevisiae Uba1 (ScUba1), and H. sapiens UBA6 (UBA6) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (C) Increasing amounts of purified H. sapiens UBA1 (UBA1) and S. cerevisiae Uba1 (ScUba1) were spotted on nitrocellulose membrane and incubated with purified pADPr chains. The retention of pADPr on the membrane was revealed using an anti-pADPr antibody. (D) Left panel: schematic representation of UBA1 with its functional domains and the six purified overlapping fragments of UBA1. Right panel: Purified MBP-UBA1 fragments resolved by PAGE and stained by Coomassie Brilliant Blue. (E) UBA1 fragments (10 pM) were spotted on nitrocellulose membrane incubated with purified pADPr chains. Immobilised pADPr was revealed using an anti-pADPr antibody. (F) Left panel: experimental scheme. MBPUBA1(571–800) was incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Eluted MBPUBA1(571–800) was resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody. IAD, inactive adenylation domain; FCCH, first catalytic cysteine half domain; AAD, active adenylation domain; SCCH, second catalytic cysteine half domain.
- Figure 5. Analysis of UBA1 and UBA6 protein sequences.
(A) The phylogenetic tree of UBA1 and UBA6 protein sequences, as inferred with MrBayes. The clustering of UBA1 orthologues is in good agreement with the phylogeny of eukaryotic subgroups. Note that evolutionary distance separates UBA1 sequences from the UBA6 out-group. (B) Alignment of the region 646–703 from the H. sapiens UBA1, and the corresponding regions of ScUba1 and H. sapiens UBA6, extracted from a larger alignment of 110 UBA1 and UBA6 sequences. The stars correspond to identical residues. The positions of nonidentical residues that were selected for mutagenesis are highlighted in yellow. (C) Structural comparison of a fragment of the Mus musculus UBA1 (H. sapiens UBA1 crystal structure is not available) and the S. cerevisiae orthologue. The seven amino acids that were chosen for mutagenesis are explicitly shown in space-filling representation. The superposition of the two proteins (bottom) highlights their close structural similarity.
- Figure S2.
Evolutionary analyses of UBA1 and UBA6 protein sequences. (A) Convergence of distributions of sampled parameter values of the evolutionary model across the four independent MrBayes runs. The effective sample size (ESS) value per parameter and run is in all cases much greater than the usually suggested minimum value of 200, indicating excellent parameter sampling. (B) Comparison of split frequencies among the four MrBayes runs. The Pearson's correlation coefficient values of 1 and the average standard deviations of split frequencies (ASDSF) that are well below 0.01 are evidence of sufficient topological convergence. (C) Evolution of cumulative average change in split frequencies during the course of each MrBayes run. Dots indicate the average change, whereas ribbons show the 100%, 95%, and 75% quantiles of the change in split frequencies. As each run independently converges on a solution, the change in split frequencies drops to nearly zero. (D) Two-dimensional projection of tree topologies explored by each MrBayes run. The high degree of similarity among plots constitutes another proof of topological convergence. (E) Heatmap of Matching Split distances (Bogdanowicz & Giaro, 2012a) among the three resulting trees and a non-phylogenetic star topology as a reference point. Overall, there are very few differences among the three trees, with the maximum likelihood ones being barely more similar to each other compared with the Bayesian tree. (F) Topological congruence among the trees produced by the three algorithms. The topology shown here is that of the MrBayes tree, as the Bayesian algorithm performed a more thorough exploration of parameter and tree space. Branches are coloured based on the taxonomic group that they belong to. Statistical support—in terms of posterior probabilities or bootstrap values—for each node across all trees is denoted with black, grey, or white circles. Nodes with black or grey circles are considered robust.
- Figure 6. UBA1 residues conserved in animals (but not in fungi) are critical for pADPr binding.
(A) Sequence logos of the alignment of the region shown in Fig 5B. The height of the letters in each position represents the degree of sequence conservation. Colours are used for differentiation among hydrophilic (blue), hydrophobic (black), or neutral amino acids (green). Error bars correspond to the Bayesian 95% confidence interval of the height. Arrows indicate the positions of the amino acids chosen for mutagenesis. These seven amino acids are not conserved between Animalia and Fungi. (B) Increasing amounts (5 to 80 pM) of purified MBPUBA1(571–800) fragments containing the indicated amino acid substitutions were spotted on a nitrocellulose membrane, incubated with purified pADPr chains, and washed. The retention of pADPr was analysed using an anti-pADPr antibody. UBA1 protein fragments used in the pADPr binding assay were revealed using an anti-MBP antibody (middle panel) or resolved by PAGE and stained with Coomassie Brilliant Blue (bottom panel). (C) U2OS cells were transfected with pAIO vector encoding an anti-UBA1 shRNA and an shRNA-resistant UBA1 cDNA encoding wild-type or mutated UBA1, as indicated, and treated with doxycyclin for 3 d. Cells were exposed to 1 μM CPT for increasing amounts of time (as indicated). Indicated proteins were detected by Western blotting.
- Figure S3.
Human UBA1 binds to pADPr chains. Left panel: Experimental scheme. Wild-type and mutated versions of MBPUBA1(571–800) were incubated with pADPr polymers, captured on an amylose resin, washed, and eluted with maltose. Right panel: Eluted MBPUBA1(571–800) proteins (wild type and mutants, as indicated) were resolved by PAGE and revealed via anti-MBP immunoblotting. Eluted pADPr was spotted on a nitrocellulose membrane and revealed using an anti-pADPr antibody.
- Figure 7. Model for the formation of ATR-signalling bodies.
Upon addition of a gapped DNA substrate in human cell-free extracts, poly(ADP-ribosyl)ation accelerates the recruitment of DNA damage signalling proteins, including the ubiquitin-activating enzyme UBA1. The coupling of pADPr-seeded protein assembly with protein ubiquitylation and protein phosphorylation prevents the formation of nonfunctional protein aggregates and promotes dedicated protein–protein interactions required for ATR signalling.
