(A, left) Chemical drawings for compounds 1, 2, and ML216. (A, right) Dose response curves from fluorescence-based DNA unwinding assays with BLM-HD. Experimental data were fitted with a four …
(Top) Chemical drawings for compounds 3 to 7. (Middle) Dose response curves from fluorescence-based DNA unwinding assays with BLM-HD. Data points are the mean of three technical replicates with …
Fitted lines are intended as visual aids only, as the data only represent values from a single preliminary experiment.
(A) Titration of BLM-HD with 2 prevents the unwinding of a forked-50mer dsDNA substrate into its component strands, as judged by native gel electrophoresis. (B) Quantification of inhibitory activity …
Experimental data were fitted with a four parameter, log(inhibitor) vs. response model with variable slope. Calculated values for IC50, Hill slope (nH) and 95% confidence intervals (95% CI) are …
(A) Schematic representation of the domain composition and respective amino acid boundaries for full-length human BLM and the two expression constructs used in this study BLM-HD and BLM-HDΔWHD. D1 …
Values for rmsd in Angstrom are shown, calculated over the indicated number of equivalent atoms. See associated key for details of colour scheme.
Values for Kd and 95% CI are given in each case. For all plots, data represent the mean of three technical replicates with error bars representing 1 SD.
Interaction network determined through by the PDBsum Generate webserver (PDBsum, 2021). Green solid lines represent hydrophobic / stacking interactions. Black dotted lines represent potential …
(A) Molecular secondary structure cartoons for the region surrounding the aromatic rich loop (ARL) of BLM-HDΔWHD (top), PDB entry 4CGZ; BLM-HD in complex with DNA (middle) and liganded complex; …
Amino acid numbering is provided for the start and end of the region compared, with the identity of key residues also provided.
Additional details for the colour scheme can be found in the associated key. The distance between the C-alpha positions of Ser729 and Asn936 is also shown, highlighting a widening of the pocket in …
(A) Molecular surface representation of BLM-HDΔWHD (left) and the liganded complex (right) highlighting the relative positions of the HRDC domain (cylindrical helices coloured in pink). The HRDC …
(A) Dose response curves from ATP-turnover assays for titration of compounds 2 and ML216 against purified recombinant BLM-HD, WRN-HD, RecQ1-HD, RecQ5-HD and UvrD respectively. Calculated values for …
Only a small movement of Thr691 would be necessary to accommodate the gamma phosphate group (as indicated by the dotted line / arrowhead). Only the D1 domain of each protein is shown in order to …
IC50 values were determined by fitting of experimental data to log (inhibitor) vs response models provided in GraphPad Prism. Data for the unwinding assay correspond to three technical replicates …
unwinding | turnover | ||
---|---|---|---|
# | Chemical drawing | IC50 [95% CI]; µM | IC50 [95% CI]; µM |
2 | ![]() | 2.2 [1.7–2.7] | 3.2 [2.3–4.0] |
3 | ![]() | 3.5 [2.4–5.2] | 5.3 [4.7–6.1] |
4 | ![]() | 6.6 [3.4–12.7] | 11.2 [8.3–15.3] |
5 | ![]() | 12.8 [4.5–36.8] | 47.86 [18.28–180.7] |
6 | ![]() | 56.9 [25.4–171.3] | 40.94 [15.9–139.8] |
7 | ![]() | No inhibition | No inhibition |
ML216 | ![]() | 4.0 [3.7–4.3] | 4.4 [4.0–4.8] |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Homo sapiens) | BLM | UniProt | P54132 | BLM_HUMAN |
Gene (Homo sapiens) | RECQL | UniProt | P46063 | RECQ1_HUMAN |
Gene (Homo sapiens) | WRN | UniProt | Q14191 | WRN_HUMAN |
Gene (Homo sapiens) | RECQL5 | UniProt | O94762 | RECQ5_HUMAN |
Gene (Escherichia coli) | uvrD | UniProt | P03018 | UVRD_ECOLI |
Strain (Escherichia coli) | BL21(DE3) | New England Biolabs | C2527I | Competent Cells |
Sequence-based reagent | ssDNA-15mer | This paper | CGTACCCGATGTGTT | |
Sequence-based reagent | ssDNA-20mer | This paper | CGTACCCGATGTGTTCGTTC | |
Sequence-based reagent | Forked-50mer: FORK A | This paper | XGAACGAACACATCGGGTACG TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT X = Black Hole Quencher 2 or none | |
Sequence-based reagent | Forked-50mer: FORK B | This paper | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT CGTACCCGATGTGTTCGTTCY Y = Tetramethylrhodamine or none | |
Recombinant DNA reagent | pET-17b | Novagen Merck Millipore | 69663 | |
Recombinant DNA reagent | pNIC28-Bsa4 | Addgene | 26103 | |
Chemical compound, drug | ML216 | Merck KGaA Caymen Chemical | SML0661 15186 | |
Commercial assay or kit | PiColorLock Gold Phosphate Detection System | Novus Biologicals | 303–0030 | |
Commercial assay or kit | DNA Unwinding Assay Kit | Inspiralis | DUKSR001 | |
Software, algorithm | BUSTER | Global Phasing | RRID:SCR_015653 | |
Software, algorithm | CCP4 | CCP4 | RRID:SCR_007255 | |
Software, algorithm | Coot | Coot | RRID:SCR_014222 | |
Software, algorithm | Fiji | Fiji | RRID:SCR_002285 | |
Software, algorithm | Phaser | Phaser | RRID:SCR_014219 | |
Software, algorithm | PHENIX | PHENIX | RRID:SCR_014224S | |
Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
Software, algorithm | XDS | XDS | RRID:SCR_015652 |
BLM-HDΔWHD + ADP | Liganded-BLM-HDΔWHD | |
---|---|---|
Data collection | ||
Space group | P21 | P1 |
Cell dimensions | ||
a, b, c (Å) | 54.28, 107.69, 55.20 | 84.69, 111.60, 132.38 |
α, β, γ (°) | 90.00, 109.31, 90.00 | 72.70, 80.13, 79.24 |
Wavelength | 0.9780 | 0.9762 |
Resolution (Å) | 51.23–1.53 (1.56–1.53) | 125.37–2.97 (3.08–2.97) |
Mn I / σI | 12.7 (1.2) | 7.8 (1.4) |
Mn I, CC1/2 | 1.00 (0.61) | 0.99 (0.57) |
Completeness (%) | 98.3 (90.3) | 98.0 (94.5) |
Redundancy | 1.9 (1.7) | 2.6 (2.7) |
Refinement | ||
Resolution (Å) | 51.23–1.53 (1.56–1.53) | 47.28–2.97 (3.07–2.96) |
No. unique reflections | 88464 (8096) | 91661 (8892) |
Rwork / Rfree | 0.19/0.21 | 0.23/0.27 |
No. atoms | ||
Macromolecules | 3870 | 24954 |
Ligands | 65 | 427 |
Solvent | 438 | 91 |
B-factors | ||
Wilson | 32.58 | 77.77 |
ADP (mean) | ||
Macromolecules | 31.00 | 95.79 |
Ligands | 40.89 | 98.56 |
Solvent | 45.69 | 53.65 |
R.m.s. deviations | ||
Bond lengths (Å) | 0.014 | 0.006 |
Bond angles (°) | 1.64 | 1.11 |
Molprobity | ||
All atom clashscore | 2.44 | 7.42 |
Ramachandran | ||
Outliers | 0.21% | 0.42% |
Allowed | 1.65% | 3.72% |
Favoured | 98.15% | 95.85% |
*Values in parentheses are for the highest resolution shell