![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
Horizontal Tabs
PDB ID |
Structure Details |
Ceftriaxone (metal site) |
|
Ceftriaxone (alternative site) |
|
Cefotaxime |
|
5Q1V |
Fragment bound at the proximal site |
5Q1U |
Fragment bound at the proximal site |
5Q1T |
Fragment bound at the proximal site |
5Q1R |
Fragment bound at the proximal site |
Fragment bound at the proximal site |
|
5Q22 |
Fragment bound at the proximal site |
5Q1K |
Fragment bound at the opposite site |
5Q1W |
Fragment bound at the opposite site |
5Q1X |
Fragment bound at the opposite site |
Experimental procedures
Expression and purification of DCLRE1A catalytic domain.
Boundaries: residues 698-1040
Vector: pFB-LIC-Bse [9,10]
Tag and additions: TEV-cleavable His6 tag at the N-terminus
(MGHHHHHHSSGVDLGTENLYFQ*SM)
Expression cell: Sf9
Note that this is a different construct from that used to generate earlier crystal structures (PDB: 5AHR and 4B87) [7].
Baculoviruses were generated using the Bac to Bac system (Life Technologies) and used to infect 2-6 L of SF9 cells in Sf-900™ II SFM medium. The cells were collected after 72 hours at 27°C, suspended in a small volume of lysis buffer (50 mM HEPES, pH 7.5, 0.5 M NaCl, 5% glycerol, 10 mM imidazole, 1 mM TCEP; protease inhibitors were added during cell resuspension but were omitted from the subsequent steps) and frozen. After thawing, the cells were diluted to ~5 volumes (v/w) of lysis buffer, then batch-bound to 5 ml of Ni-loaded chelating sepharose beads (GE) for 1 hour. The beads were washed with lysis buffer containing 30 mM imidazole, and the protein was eluted with lysis buffer containing 300 mM imidazole.
The tag was cleaved by incubating the protein overnight at 4°C with TEV protease (1/40 (w/w)) in a dialysis tube placed in 1 L dialysis buffer (50 mM HEPES, 0.5 M NaCl, 5% glycerol and 1 mM TCEP). The protein was then passed through a 1-mL Ni-sepharose column, and the flowthrough fractions were collected. The protein was concentrated on a centrifugal concentrator (Centricon, MWCO 30 kDa) before loading on a Superdex S200 HR 16/60 gel filtration column in dialysis buffer, and separated at 1.2 mL/min. fractions containing purified DCLRE1A protein were identified by SDS-PAGE, pooled and concentrated to 9-10 mg/mL.
Protein crystallization and compound soaking
Protein crystallization was performed by vapour diffusion in sitting drops at 4°. A protein solution at 9-10 mg/mL was mixed at with an equal volume crystallization solution containing 30% PEG 1000, 0.1M MIB pH 6.0 (MIB is Sodium malonate dibasic monohydrate, Imidazole, Boric acid). The crystals contained malonate bound to the active site. For fragment screening, concentrated solutions (0.5 M) of the fragments in DMSO were added to the crystallization drops using an Echo dispenser, up to 10% of the drop volume, at the Diamond light source XChem facility. Crystals were harvested after 1-4 hour of incubation, flash-cooled in liquid N2 without additional cryoprotectant. For the cephalosporin compound soaks, the crystals were first incubated overnight in malonate-free liquor (30% PEG, 0.1 M HEPES, pH 7.0) to remove the malonate at the active site; compounds were subsequently soaked overnight at a final concentration of approximately 20 mM before harvesting and cryo-cooling. Data was collected in Diamond light source beamline I04-1 (Fragments), I04 and ESRF beamline BM30B.
Activity assay
Real-time Fluorescence Assays: Real-time fluorescence assays were performed as described [1, 8] using the 20-mer oligonucleotide:
5’-phosphate-A-[fluorescein-T]-AATTTGATCA [BHQ1-T] CTATTAT
In this oligonucleotide, fluorescence from the fluorescein linked at position 2 is quenched by the BHQ1 group linked to position 13. When the first two nucleotides are hydrolysed, the unquenched fluorescence of the free fluorescein-TMP can be detected in real time (excitation at 495 nm, emission at 525 nm).
Reactions were carried out in black 384-well microplates, and measurements were made using a SpectraMax M2e fluorescent plate reader in fluorescent top read mode, with SoftMaxPro software (Molecular Devices, Sunnyvale, CA, USA) to control the settings. Reactions were performed in a total volume of 15 μL in nuclease buffer (20 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.05% Triton-X100, 0.1 mg/mL BSA, 5% glycerol) with varying concentrations of compound (for IC50 determinations) or DNA substrate (for kinetics assays) and 0.24 nM DCLRE1A (the Km for ssDNA oligo was < 5 nM). Each reaction was started by the addition of DCLRE1A, and the fluorescein emission spectra measured (ex. 495 nm, em. 525 nm and cutoff at 515 nm) with six readings taken at 7 s intervals for 6 min. The fluorescence intensity of each well was plotted against time, and the rate of increase was determined, plotted against compound or substrate concentration and fitted to a log(inhibitor)- response or Michaelis–Menten curve on Prism software (GraphPad Software, Inc., La Jolla, CA, USA) to determine IC50 or Km and Vmax.
For radioactive gel-based assays, similar reaction conditions were used, with substrate DNAs labelled at the 3’ end with α-[32P]-dATP and terminal deoxynucleotidyl transferase. Reactions were stopped by boiling in sample buffer (95% formamide, 10 mM EDTA, bromophenol blue and xylene cyanol) and analysed by electrophoresis on 20% polyacrylamide, 0.5 x TBE and 7M urea.
Gene Knockouts
CRISPR/Cas9 knockout of DCLRE1A has been performed in the McHugh lab. Contact Prof. Peter McHugh, Oxford (peter.mchugh@imm.ox.ac.uk) for collaborative opportunities.
Antibodies
We are not aware of antibodies that can detect the native levels of DCLRE1A in wester/immunofluorescence.
IMPORTANT: Please note that the existence of small molecules within this TEP may only indicate that chemical matter can bind to the protein in a functionally relevant pocket. As such these molecules should not be used as tools for functional studies of the protein unless otherwise stated as they are not potent enough and not characterised enough to be used in cellular studies. A TEP's small molecule ligands are intended to be used as the basis for future chemistry optimisation to increase potency and selectivity and yield a chemical probe or lead series.
PDB ID |
Compound structure |
Fragments in proximal pocket |
|
5Q1V |
![]() |
5Q1U |
![]() |
5Q1T |
![]() |
5Q1R |
![]() |
![]() |
|
5Q22 |
![]() |
Fragments in opposite pocket |
|
5Q1K |
![]() |
5Q1W |
![]() |
5Q1X |
![]() |
Cephalosporin inhibitors |
|
|
|
5NZY: Cefotaxime |
|
- Sengerova, B., Allerston, C. K., Abu, M., Lee, S. Y., Hartley, J., Kiakos, K., Schofield, C. J., Hartley, J. A., Gileadi, O., and McHugh, P. J. (2012) Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases. J Biol Chem 287, 26254-26267
- Wang, A. T., Sengerova, B., Cattell, E., Inagawa, T., Hartley, J. M., Kiakos, K., Burgess-Brown, N. A., Swift, L. P., Enzlin, J. H., Schofield, C. J., Gileadi, O., Hartley, J. A., and McHugh, P. J. (2011) Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair. Genes Dev 25, 1859-1870
- Cattell, E., Sengerova, B., and McHugh, P. J. (2010) The SNM1/Pso2 family of ICL repair nucleases: from yeast to man. Environ Mol Mutagen 51, 635-645
- Hazrati, A., Ramis-Castelltort, M., Sarkar, S., Barber, L. J., Schofield, C. J., Hartley, J. A., and McHugh, P. J. (2008) Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants. DNA Repair (Amst) 7, 230-238
- Iyama, T., Lee, S. Y., Berquist, B. R., Gileadi, O., Bohr, V. A., Seidman, M. M., McHugh, P. J., and Wilson, D. M., 3rd. (2015) CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. Nucleic acids research 43, 247-258
- Yang, K., Moldovan, G. L., and D'Andrea, A. D. (2010) RAD18-dependent recruitment of SNM1A to DNA repair complexes by a ubiquitin-binding zinc finger. J Biol Chem 285, 19085-19091
- Allerston, C. K., Lee, S. Y., Newman, J. A., Schofield, C. J., McHugh, P. J., and Gileadi, O. (2015) The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities. Nucleic acids research 43, 11047-11060
- Lee, S. Y., Brem, J., Pettinati, I., Claridge, T. D., Gileadi, O., Schofield, C. J., and McHugh, P. J. (2016) Cephalosporins inhibit human metallo beta-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo. Chem Commun (Camb) 52, 6727-6730
We respectfully request that this document is cited using the DOI value as given above if the content is used in your work.