Journal: eLife

Article Title: Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

doi: 10.7554/eLife.42144

Figure Lengend Snippet: ( A ) Actin and myosin II stress fiber formation in non-muscle cells. Actin stress fibers are formed via the Arp2/3 complex and the formin mDia1. NMIIA is the predominant isoform at the leading edge of non-muscle cells, and stress fiber formation is NMIIA dependent. Non-muscle cells display robust retrograde flow of actin stress fibers and display rapid turnover. Large NMIIA stacks are formed via growth and expansion of smaller NMIIA filaments. Citations leading to this model are presented in the cartoon. ( B ) Model of actin and myosin II stress fiber formation in human cardiomyocytes. Sarcomeres are templated by Muscle Stress Fibers (MSFs). MSFs do not require the Arp2/3 complex, and require the formin FHOD3. MSFs display slow retrograde flow compared with non-muscle stress fibers. Both NMIIA and NMIIB are localized to the edge of hiCMs, and display prominent NMII co-filaments. NMIIB-βCMII co-filaments are also present with MSFs. Large βCMII filament stacks form via concatenation and stitching of individual βCMII filaments.

Article Snippet: The following dataset was generated: Fenix AM Burnette DT 2018 Cardiomyocyte mRNA Content NCBI Gene Expression Omnibus GSE119743

Techniques:

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) The domain architecture of C. crescentus ParB: the NTD (dark green), the central DNA-binding domain (DBD, dark green), the C-terminal domain (CTD, faded green), and a linker that connects the DBD and the CTD together. The ParB∆CTD variant that was used for crystallization lacks the CTD (faded green). ( B , left panel) Co-crystal structure of two C. crescentus ParB∆CTD monomers (dark green and gray) bound to a 22 bp parS DNA. The nucleotide sequence of the 22 bp parS is shown below the co-crystal structure, the core parS sequence is highlighted in bold, and each parS half-site is denoted by an arrow. The position of residue L224 is also indicated. (Right panel) The structure of a ParB∆CTD subunit bound to a parS half site with key features highlighted. ( C ) Superimposition of C. crescentus ParB∆CTD subunits shows two different orientations of the NTD. The arrow above each subunit shows the direction each NTD is projecting towards. ( D ) A top-down view of the superimposition of ParB∆CTD subunits shows their NTDs orienting ~80° apart from each other.

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Binding Assay, Variant Assay, Crystallization Assay, Sequencing, Residue

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) The ASU contains four copies of the C. crescentus ParB∆CTD monomers (chains A, B, C, and D) and two copies of the full-size parS DNA. ( B ) Superimposition of the chain C-D- parS complex (dark green) to the chain A-B- parS complex (cyan) shows that the two complexes in the ASU are structurally similar (root-mean-square deviation [RMSD] = 1.59 Å).

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques:

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) A side view of the superimposition between C. crescentus ParB∆CTD chain C (gray), chain D (dark green), and H. pylori ParB∆CTD (golden) shows the three distinct orientations of the N-terminal domain (NTD). ( B ) A top view of the superimposition between C. crescentus ParB∆CTD chain C, chain D, and H. pylori ParB∆CTD. The parS DNA, DNA-binding domain (DBD), and helix α4 are omitted for clarity.

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Binding Assay

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) Structural changes between C. crescentus ParB∆CTD- parS and ParB∆CTD-CTPɣS structures. Helices α3 and α4 are shown in light blue. The arrows next to the N-terminal domain (NTD) (residues 44–121) and the DBD (residues 161–221) show the direction that these domains rotate towards in the nucleotide-bound state. ( B ) Superimposing the C. crescentus ParB∆CTD-CTPɣS structure onto parS DNA shows DNA-recognition helices (α6 and α6′, magenta) positioning away from the two consecutive major grooves of parS , and helices α8–α9 and α8′–α9′ at the DBD (dashed box) clashing with parS DNA.

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques:

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) A schematic diagram of C. crescentus ParB showing the position of Q35 (at the N-terminal domain [NTD]), L224 (at the DBD), and I304 (at the CTD) that were substituted either individually or in combinations for cysteine. ( B ) Denaturing polyacrylamide gel analysis of bismaleimidoethane (BMOE) crosslinking products of 8 µM single-cysteine ParB (Q35C/L224C/I304C) variant ±0.5 µM 22 bp parS DNA ±1 mM CTP. X indicates a crosslinked form of ParB. Quantification of the crosslinked (X) fraction is shown below each representative gel image. Error bars represent SD from three replicates. ( C , left panel) Denaturing polyacrylamide gel analysis of BMOE crosslinking products of 8 µM dual-cysteine ParB (Q35C I304C) variant ±0.5 µM DNA ±1 mM CTP. Different DNA were employed in crosslinking reactions: a linear 22 bp parS DNA (22 bp parS lin), a circular 3 kb parS plasmid (3 kb parS cir), and a circular 3 kb scrambled parS plasmid (3 kb nonS cir). The high molecular weight (HMW) smear near the top of the polyacrylamide gel is marked with a solid line and an asterisk (lane 7). When the crosslinking reaction was post-treated with a non-specific DNA nuclease, Benzonase, the HMW smear was no longer observed (dashed line and asterisk, lane 8). The polyacrylamide gel was also stained with a DNA dye, Sybr Green (SYBR), and only the top section of the gel is shown. Small 22 bp parS DNA duplex migrated out of the gel, thus was not observed near the top of the Sybr-stained gel. A schematic diagram of a dual-cysteine C. crescentus ParB dimer is also shown. (Right panel) Agarose gel analysis of BMOE crosslinking products. A subset of crosslinking reactions (lanes 6, 7, and 9–12) were loaded and resolved on 1% agarose gel. The gel was subsequently stained with Sybr Green for DNA. Shifted gel bands are marked with a solid line and an asterisk. ( D ) Same as panel ( C ) but another dual-cysteine variant, ParB (L224C I304C) was employed instead. Figure 5—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Variant Assay, Plasmid Preparation, High Molecular Weight, Staining, SYBR Green Assay, Agarose Gel Electrophoresis

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: Denaturing polyacrylamide gel analysis of bismaleimidoethane (BMOE) crosslinking products of 8 µM dual-cysteine ParB variants + 0.5 µM (circular/linearized) parS -containing plasmid DNA ±1 mM CTP. Purified ParB (Q35C I304C), ParB (L224C I304C), and ParB (Q35C L224C) were used for reactions in lanes 1–4, 5–8, and 9–12, respectively. Different DNA were employed in crosslinking reactions: a circular 3 kb parS plasmid (3 kb parS cir) and a 3 kb parS plasmid that had been linearized at an unique HindIII site by HindIII restriction enzyme (3 kb parS linear). The HMW smear near the top of the polyacrylamide gel was observed when ParB (Q35C I304C) or ParB (L224C I304C) was incubated with CTP and a circular parS plasmid (lanes 2 and 6, solid lines and asterisks), but not when a linearized parS plasmid was used (lanes 4 and 8, dashed lines and asterisks) or when CTP was omitted (lanes 1 and 5). ParB (Q35C L224C) did not produce a HMW smear even in the presence of CTP and a circular parS plasmid (see also ). Crosslinking reactions were also loaded and resolved on a 1% agarose gel. The gel was subsequently stained with Sybr Green for DNA. Shifted gel bands are marked with a solid line and an asterisk. Figure 5—figure supplement 1—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Plasmid Preparation, Purification, Incubation, Agarose Gel Electrophoresis, Staining, SYBR Green Assay

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: (Left panel) Denaturing polyacrylamide gel analysis of bismaleimidoethane (BMOE) crosslinking products of 8 µM dual-cysteine ParB (Q35C L224C) variant ±0.5 µM DNA ±1 mM CTP. Different DNA were employed in crosslinking reactions: a linear 22 bp parS DNA (22 bp parS lin), a circular 3 kb parS plasmid (3 kb parS cir), and a circular 3 kb scrambled parS plasmid (3 kb nonS cir). The HMW smear near the top of the polyacrylamide gel was not observed, with or without a Benzonase post-treatment (dashed lines and asterisks, lanes 7 and 8). The polyacrylamide gel was also stained with a DNA-dye, Sybr Green (SYBR), and only the top section of the gel is shown. Small 22 bp parS DNA duplex migrated out of the gel, thus was not observed near the top of the Sybr-stained gel. A schematic diagram of a dual-cysteine C. crescentus ParB dimer is also shown. (Right panel) Agarose gel analysis of BMOE crosslinking products. A subset of crosslinking reactions (lanes 6, 7, and 9–12) were loaded and resolved on 1% agarose gel. The gel was subsequently stained with Sybr Green for DNA. Shifted gel bands are marked with a solid line and an asterisk. Figure 5—figure supplement 2—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Variant Assay, Plasmid Preparation, Staining, SYBR Green Assay, Agarose Gel Electrophoresis

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) A post-crosslinking treatment of a ParB (L224C I304C)-TEV variant with TEV protease eliminated the HMW smear. A dual-cysteine ParB variant was engineered with a TEV protease cleavage site in the DNA-binding domain–C-terminal domain (DBD-CTD) linker (see the schematic diagram). This ParB (L224C I304C)-TEV variant was purified and used in a bismaleimidoethane (BMOE) crosslinking reactions in the presence or absence of 1 mM cytidine triphosphate (CTP) and 0.5 µM DNA. Different DNA were employed in crosslinking reactions: a linear 22 bp parS DNA (22 bp parS lin), a circular 3 kb parS plasmid (3 kb parS cir), and a circular 3 kb scrambled parS plasmid (3 kb nonS cir). In contrast to lane 7, the HMW smear near the top of the polyacrylamide gel was no longer observed when TEV was added after the crosslinking reactions was quenched (lane 8, dashed line and asterisk). ( B ) Double-crosslinked ParB (L224C I304C) dimer is the major protein species in the HMW smear. Gel slices encompassing the HMW smear were crushed and soaked in a Benzonase-supplemented buffer to release bound proteins. The released protein was analyzed on a denaturing polyacrylamide gel (lane 9). The solid arrow indicates the position of a double-crosslinked ParB (L224C I304C). Figure 5—figure supplement 3—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Variant Assay, Binding Assay, Purification, Plasmid Preparation

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) A schematic diagram of the biolayer interferometry (BLI) assay. A dual biotin-labeled 170 bp parS DNA was tethered to the streptavidin (SA)-coated probe to create a DNA substrate where both ends were blocked (a closed DNA substrate). BLI assay monitors wavelength shifts (nm) resulting from changes in the optical thickness of the sensor surface during association or dissociation of the analyte. In the absence of cytidine triphosphate (CTP), ParB (green) nucleates at parS only. In the presence of CTP (orange), ParB slides on and entraps DNA to accumulate on the closed DNA substrate, thus giving rise to an elevated BLI response. ( B ) A prolonged (30 min) pre-incubation of ParB (Q35C) with CTPɣS eliminated most of the ParB binding and accumulation on DNA. It was shown previously that CTPɣS, in the absence of parS DNA, gradually converted apo-ParB from an open to a closed protein clamp ( ; ). If not already bound on DNA, the now inaccessible DNA-binding domain of ParB cannot bind parS to nucleate and to subsequently slide to accumulate on DNA. ( C ) An irreversible closing of ParB clamps, by bismaleimidoethane (BMOE)-mediated crosslinking a closed form of ParB (Q35C), also eliminated most of the ParB binding and accumulation on DNA. Before BLI experiment, purified ParB (Q35C) was incubated with CTPɣS for 30 min, then 1 mM BMOE was added to crosslink the N-terminal domains (NTDs) of ParB (Q35C) together. The crosslinking reaction was quenched with 1 mM DTT and ParB (Q35C) was purified away from excess BMOE and DTT using a Zeba buffer exchange column (see Materials and methods). Approximately 70% ParB (Q35C) was in a crosslinked form . CTPɣS was required to convert apo-ParB (Q35C) to a closed clamp form before crosslinking. Without CTPɣS, most apo-ParB (Q35C) is in an open clamp form . For all BLI reactions in this figure, 5 µM of ParB (Q35C) and 1 mM CTP/CTPɣS were used. Each BLI experiment was triplicated and a representative sensorgram is presented. Figure 5—figure supplement 4—source data 1. Data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Labeling, Incubation, Binding Assay, Purification, Buffer Exchange

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: Eleven residues at C-motif and P-motifs 1–3 were individually substituted for alanine or glycine. ( A ) Membrane-spotting assay of ParB variants. CTP binding was monitored by membrane-spotting assay using radiolabeled CTP α-P 32 . The bulls-eye staining indicates CTP binding due to a more rapid immobilization of protein-ligand complexes compared to free ligands. All reactions contained various concentration of purified ParB, 5 nM radiolabeled CTP α-P 32 , 30 µM unlabeled CTP, and 1.5 µM 22 bp parS DNA. The bound fractions were quantified, and error bars represent SD from three replicates. All the reactions were spotted on the same membrane, the radiograph was rearranged solely for presentation purposes. ( B ) Inorganic phosphate release assay of ParB variants. The CTPase rates were measured at increasing concentration of CTP. All reactions contained 1 µM purified ParB variant, 0.5 µM 22 bp parS DNA, and an increasing concentration of CTP. ( C ) Bismaleimidoethane (BMOE) crosslinking assay of ParB variants. A second set of alanine scanning ParB variants, which harbor an additional Q35C substitution at the N-terminal domain (NTD), were also constructed and subsequently used in BMOE crosslinking experiments. Purified ParB variants (8 µM) were preincubated with 0.5 µM 22 bp parS DNA and an increasing concentration of CTP for 5 min before BMOE was added. Crosslinking products were resolved on a 12% denaturing polyacrylamide gel and the crosslinked fractions were quantified (see also for representation images). Error bars represent SD from three replicates. ( D ) Biolayer interferometry (BLI) assay of ParB variants. BLI analysis of the interaction between a premix of 1 µM ParB variant ± an increasing concentration of CTP and a 170 bp closed parS DNA substrate. See also for a schematic diagram of the BLI setup and for representative BLI sensorgrams. BLI signal at the end of the association phase (± SD from three replicates) was plotted against CTP concentrations. Figure 6—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Membrane, Spotting Assay, Binding Assay, Staining, Concentration Assay, Purification, Phosphate Release Assay, Variant Assay, Construct

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: Purified ParB variants (8 µM) were preincubated with 0.5 µM 22 bp parS DNA, and an increasing concentration of cytidine triphosphate (CTP) for 5 min before bismaleimidoethane (BMOE) was added. Crosslinking products were resolved on a 12% denaturing polyacrylamide gel and the crosslinked fractions (X) were quantified. Error bars represent SD from three replicates. Figure 6—figure supplement 1—source data 1. Original files, annotation of the full raw gels, and data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Purification, Concentration Assay

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: Representative BLI sensorgrams of the interactions between ParB variants and the closed DNA substrate in the presence of an increasing concentration of cytidine triphosphate (CTP). The BLI probe with tethered parS DNA substrate was dipped into a buffer-only solution (0–30 s), then to a premix of 1 µM ParB ± an increasing concentration of CTP (30–150 s: association phase), and finally returned to a buffer-only solution (150–270 s: dissociation phase). Each BLI experiment was triplicated and a representative sensorgram is presented. Figure 6—figure supplement 2—source data 1. Data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Concentration Assay

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ( A ) Biolayer interferometry (BLI) analysis of the interaction between a premix of 1 µM C. crescentus ParB (WT) or ParB (E102A) + 1 mM cytidine triphosphate (CTP) and 170 bp dual biotin-labeled parS DNA. For the dissociation phase, the probe was returned to a low-salt buffer that contains 100 mM NaCl (solid black or red lines) or to a high-salt buffer that contains 1 M NaCl (dashed black or red lines). The schematic diagram of the BLI probe shows a closed parS DNA substrate due to the interactions between a dual biotin-labeled DNA and the streptavidin (SA)-coated probe surface. ( B ) BLI analysis of the interaction between a premix of 1 µM C . crescentus ParB (WT) or ParB (E102A) + 1 mM CTP (solid lines) or –1 mM CTP (dashed lines) and 170 bp dual biotin-labeled parS DNA. ( C ) Same as panel ( B ) but immobilized DNA fragments have been restricted with BamHI before BLI analysis. Figure 7—source data 1. Data used to generate .

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Labeling

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: ParB (dark green) consists of three domains: an N-terminal CTP-binding domain (NTD), a central parS DNA-binding domain (DBD), a C-terminal dimerization domain (CTD), and a 20 amino acid linker that connects the DBD and the CTD together. Nucleating ParB is an open clamp, in which parS DNA is captured at the DBD (the DNA-gate). Upon binding CTP (orange), the NTD self-dimerizes to close the NTD-gate of the clamp. CTP-binding and the exchange of helices α4 and α4′ (blue) stabilize this closed conformation. The DBD also move closer together to close the DNA-gate, potentially driving parS DNA into a compartment between the DNA-gate and the C-terminal domain. In the nucleotide-bound state, the DBD and the DNA-recognition helices (α6 and α6′, magenta) are incompatible with DNA binding. CTP hydrolysis and/or the release of hydrolytic products (CDP and inorganic phosphate Pi) may reopen the gates to release DNA. Substitutions that affect key steps in the CTP biding/hydrolysis cycle are also indicated on the schematic diagram.

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: Binding Assay

Journal: eLife

Article Title: A CTP-dependent gating mechanism enables ParB spreading on DNA

doi: 10.7554/eLife.69676

Figure Lengend Snippet: X-ray data collection and processing statistics.

Article Snippet: The following dataset was generated: Le TBK 2021 A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus NCBI Gene Expression Omnibus GSE168968 Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2020 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed to the centromeric parS site RCSB Protein Data Bank 6T1F Jalal ASB Pastrana CL Tran NT Stevenson CEM Lawson DM Moreno-Herrero F Le TBK 2021 Crystal structure of the C-terminally truncated chromosome-partitioning protein ParB from Caulobacter crescentus complexed with CTP-gamma-S RCSB Protein Data Bank 7BM8

Techniques: