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Protein Expression and Purification
Construct: DCLRE1CA-c401
DCLRE1C catalytic domain
Boundaries: residues 1-362
Vector: pFB-6HZB (Genbank: JF682518.1)
Tag and additions: TEV-cleavable His6 tag at the N-terminus with the addition of Z-basic tag
Expression cell: Sf9
Protein sequence (Tag sequence is underlined, and the TEV protease cleavage site is indicated with *):
MGHHHHHHSSGVDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQPKGTENLYFQ*SMSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRAPTLKRRLECSLKVYLYCSPVTKELLLTSPKYRFWKKRIISIEIETPTQISLVDEASGEKEEIVVTLLPAGHCPGSVMFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSREECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPEILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCRS
Predicted mass: 50730.3
Protein after tag cleavage:
SMSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRAPTLKRRLECSLKVYLYCSPVTKELLLTSPKYRFWKKRIISIEIETPTQISLVDEASGEKEEIVVTLLPAGHCPGSVMFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSREECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPEILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCRS
Predicted mass: 41715.2
DNA sequence (ORF):
ATGGGCCACCATCATCATCATCATTCTTCTGGTGTGGATAACAAGTTCAACAAGGAGCGTCGAAGAGCTCGCCGTGAAATTCGCCATCTGCCGAACCTGAACCGCGAACAGCGTCGCGCATTTATTCGCAGCCTGCGCGATGATCCGAGCCAGAGCGCGAACCTGCTGGCGGAAGCGAAGAAGCTGAACGATGCGCAGCCGAAGGGTACAGAGAACCTGTACTTCCAATCCATGAGTTCTTTCGAGGGGCAGATGGCCGAGTATCCAACTATCTCCATAGACCGCTTCGATAGGGAGAACCTGAGGGCCCGCGCCTACTTCCTGTCCCACTGCCACAAAGATCACATGAAAGGATTAAGAGCCCCTACCTTGAAAAGAAGGTTGGAGTGCAGCTTGAAGGTTTATCTATACTGTTCACCTGTGACTAAGGAGTTGTTGTTAACGAGCCCGAAATACAGATTTTGGAAGAAACGAATTATATCTATTGAAATCGAGACTCCTACCCAGATATCTTTAGTGGATGAAGCATCAGGAGAGAAGGAAGAGATTGTTGTGACTCTCTTACCAGCTGGTCACTGTCCGGGATCAGTTATGTTTTTATTTCAGGGCAATAATGGAACTGTCCTGTACACAGGAGACTTCAGATTGGCGCAAGGAGAAGCTGCTAGAATGGAGCTTCTGCACTCCGGGGGCAGAGTCAAAGACATCCAAAGTGTATATTTGGATACTACGTTCTGTGATCCAAGATTTTACCAAATTCCAAGTCGGGAGGAGTGTTTAAGTGGAGTCTTAGAGCTGGTCCGAAGCTGGATCACTCGGAGCCCGTACCATGTTGTGTGGCTGAACTGCAAAGCGGCTTATGGCTATGAATATTTGTTCACCAACCTTAGTGAAGAATTAGGAGTCCAGGTTCATGTGAATAAGCTAGACATGTTTAGGAACATGCCTGAGATCCTTCATCATCTCACAACAGACCGCAACACTCAGATCCATGCATGCCGGCATCCCAAGGCAGAGGAATATTTTCAGTGGAGCAAATTACCCTGTGGAATTACTTCCAGAAATAGAATTCCACTCCACATAATCAGCATTAAGCCATCCACCATGTGGTTTGGAGAAAGGAGCAGAAAAACAAATGTAATTGTGAGGACTGGAGAGAGTTCATACAGAGCTTGTTTTTCTTTTCACTCCTCCTACAGTGAGATTAAAGATTTCTTGAGCTACCTCTGTCCTGTGAACGCATATCCAAATGTCATTCCAGTTGGCACAACTATGGATAAAGTTGTCGAAATCTTAAAGCCTTTATGCCGGTCTTGA
Expression and purification of DCLRE1C catalytic domain (aa 1-362)
Baculovirus generation was performed as previously described in Allerston et.al (16). The proteins were expressed in SF9 cell at 2 x 106 cells/ mL infected with 1.5 mL of P2 virus. Infected Sf9 cells were harvested 70 h at 27 °C after infection by centrifugation at 900g for 20 min. The cell pellet was resuspended in 30 mL/L lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% v/v glycerol and 1 mM TCEP), snap frozen in liquid nitrogen, and stored at −80 °C for later use.
Thawed cell aliquots were lysed by sonication. The lysate was clarified by centrifugation at 40,000g for 30 min, and the supernatant was passed through a 0.80 μm filter (Millipore) before being loaded onto an equilibrated (lysis buffer) immobilised metal affinity chromatography column (IMAC) (Ni-NTA superflow Cartridge, Qiagen).
The immobilised protein was washed with lysis buffer and eluted using a linear gradient of elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% v/v glycerol and 1 mM TCEP). The protein containing fractions were pooled and passed through an ion exchange column (HiTrap® SP FF GE Healthcare Life Sciences) pre-equilibrated in the SP buffer A (25 mM HEPES pH 7.5, 300 mM NaCl, 5% v/v glycerol and 1 mM TCEP). The protein was eluted using a linear gradient of SP buffer B (SP buffer A with 1 M NaCl).
The protein-containing fractions were pooled and dialysed overnight at 4 °C in SP buffer A and supplemented with recombinant tobacco etch virus (TEV) protease for cleavage of the 6His-ZB tag. The protein was subsequently loaded into an ion exchange column (HiTrap® SP FF GE Healthcare Life Sciences) pre-equilibrated in the SP buffer A to remove 6His-ZB tag and un-cleaved protein. The protein was eluted using a linear gradient of SP buffer B.
Artemis-containing fractions from the SP column elution were combined and concentrated down to 1 mL using a 30 kDa MWCO Amicon® centrifugal concentrator. The protein was then loaded on to a Superdex 75 increase 10/300 GL equilibrated with SEC buffer (25 mM HEPES pH 7.5, 300 mM NaCl, 5% v/v glycerol, 2 mM TCEP).
DCLRE1C Full length
Boundaries: residues 1-692
Vector: pFB-CT10HF (Addgene http://www.addgene.org/39191/ )
Tag and additions: TEV-cleavable His10 tag at the C-terminus with the addition of FLAG tag
Expression cell: Sf9
Protein sequence (Tag sequence is underlined, *:TEV protease cleavage site):
MSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRAPTLKRRLECSLKVYLYCSPVTKELLLTSPKYRFWKKRIISIEIETPTQISLVDEASGEKEEIVVTLLPAGHCPGSVMFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSREECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPEILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCRSSQSTEPKYKPLGKLKRARTVHRDSEEEDDYLFDDPLPIPLRHKVPYPETFHPEVFSMTAVSEKQPEKLRQTPGCCRAECMQSSRFTNFVDCEESNSESEEEVGIPASLQGDLGSVLHLQKADGDVPQWEVFFKRNDEITDESLENFPSSTVAGGSQSPKLFSDSDGESTHISSQNSSQSTHITEQGSQGWDSQSDTVLLSSQERNSGDITSLDKADYRPTIKENIPASLMEQNVICPKDTYSDLKSRDKDVTIVPSTGEPTTLSSETHIPEEKSLLNLSTNADSQSSSDFEVPSTPEAELPKREHLQYLYEKLATGESIAVKKRKCSLLDTAENLYFQ*SHHHHHHHHHHDYKDDDDK
Expected mass: 81756
Protein sequence (after tag cleavage):
MSSFEGQMAEYPTISIDRFDRENLRARAYFLSHCHKDHMKGLRAPTLKRRLECSLKVYLYCSPVTKELLLTSPKYRFWKKRIISIEIETPTQISLVDEASGEKEEIVVTLLPAGHCPGSVMFLFQGNNGTVLYTGDFRLAQGEAARMELLHSGGRVKDIQSVYLDTTFCDPRFYQIPSREECLSGVLELVRSWITRSPYHVVWLNCKAAYGYEYLFTNLSEELGVQVHVNKLDMFRNMPEILHHLTTDRNTQIHACRHPKAEEYFQWSKLPCGITSRNRIPLHIISIKPSTMWFGERSRKTNVIVRTGESSYRACFSFHSSYSEIKDFLSYLCPVNAYPNVIPVGTTMDKVVEILKPLCRSSQSTEPKYKPLGKLKRARTVHRDSEEEDDYLFDDPLPIPLRHKVPYPETFHPEVFSMTAVSEKQPEKLRQTPGCCRAECMQSSRFTNFVDCEESNSESEEEVGIPASLQGDLGSVLHLQKADGDVPQWEVFFKRNDEITDESLENFPSSTVAGGSQSPKLFSDSDGESTHISSQNSSQSTHITEQGSQGWDSQSDTVLLSSQERNSGDITSLDKADYRPTIKENIPASLMEQNVICPKDTYSDLKSRDKDVTIVPSTGEPTTLSSETHIPEEKSLLNLSTNADSQSSSDFEVPSTPEAELPKREHLQYLYEKLATGESIAVKKRKCSLLDTAENLYFQ
Predicted mass: 79302.5
DNA sequence (ORF):
ATGAGTTCTTTCGAGGGGCAGATGGCCGAGTATCCAACTATCTCCATAGACCGCTTCGATAGGGAGAACCTGAGGGCCCGCGCCTACTTCCTGTCCCACTGCCACAAAGATCACATGAAAGGATTAAGAGCCCCTACCTTGAAAAGAAGGTTGGAGTGCAGCTTGAAGGTTTATCTATACTGTTCACCTGTGACTAAGGAGTTGTTGTTAACGAGCCCGAAATACAGATTTTGGAAGAAACGAATTATATCTATTGAAATCGAGACTCCTACCCAGATATCTTTAGTGGATGAAGCATCAGGAGAGAAGGAAGAGATTGTTGTGACTCTCTTACCAGCTGGTCACTGTCCGGGATCAGTTATGTTTTTATTTCAGGGCAATAATGGAACTGTCCTGTACACAGGAGACTTCAGATTGGCGCAAGGAGAAGCTGCTAGAATGGAGCTTCTGCACTCCGGGGGCAGAGTCAAAGACATCCAAAGTGTATATTTGGATACTACGTTCTGTGATCCAAGATTTTACCAAATTCCAAGTCGGGAGGAGTGTTTAAGTGGAGTCTTAGAGCTGGTCCGAAGCTGGATCACTCGGAGCCCGTACCATGTTGTGTGGCTGAACTGCAAAGCGGCTTATGGCTATGAATATCTGTTCACCAACCTTAGTGAAGAATTAGGAGTCCAGGTTCATGTGAATAAGCTAGACATGTTTAGGAACATGCCTGAGATCCTTCATCATCTCACAACAGACCGCAACACTCAGATCCATGCATGCCGGCATCCCAAGGCAGAGGAATATTTTCAGTGGAGCAAATTACCCTGTGGAATTACTTCCAGAAATAGAATTCCACTCCACATAATCAGCATTAAGCCATCCACCATGTGGTTTGGAGAAAGGAGCAGAAAAACAAATGTAATTGTGAGGACTGGAGAGAGTTCATACAGAGCTTGTTTTTCTTTTCACTCCTCCTACAGTGAGATTAAAGATTTCTTGAGCTACCTCTGTCCTGTGAACGCATATCCAAATGTCATTCCAGTTGGCACAACTATGGATAAAGTTGTCGAAATCTTAAAGCCTTTATGCCGGTCTTCCCAAAGTACGGAGCCAAAGTATAAACCACTGGGAAAACTGAAGAGAGCTAGAACAGTTCACCGAGACTCAGAGGAGGAAGATGACTATCTCTTTGATGATCCTCTGCCAATACCTTTAAGGCACAAAGTTCCATACCCGGAAACTTTTCACCCTGAGGTATTTTCAATGACTGCAGTATCAGAAAAGCAGCCTGAAAAACTGAGACAAACCCCAGGATGCTGCAGAGCAGAGTGTATGCAGAGCTCTCGTTTCACAAACTTTGTAGATTGTGAAGAATCCAACAGTGAAAGTGAAGAAGAAGTAGGAATCCCAGCTTCACTGCAAGGAGATCTGGGCTCTGTACTTCACCTGCAAAAGGCTGATGGGGATGTACCCCAGTGGGAAGTATTCTTTAAAAGAAATGATGAAATCACAGATGAGAGTTTGGAAAACTTCCCTTCCTCCACAGTGGCAGGGGGATCTCAGTCACCAAAGCTTTTCAGTGACTCTGATGGAGAATCAACTCACATCTCCTCCCAGAATTCTTCCCAGTCAACACACATAACAGAACAAGGAAGTCAAGGCTGGGACAGCCAATCTGATACTGTTTTGTTATCTTCCCAAGAGAGAAACAGTGGGGATATTACTTCCTTGGACAAAGCTGACTACAGACCAACAATCAAAGAGAATATTCCTGCCTCTCTCATGGAACAAAATGTAATTTGCCCAAAGGATACTTACTCTGATTTGAAAAGCAGAGATAAAGATGTGACAATAGTTCCTAGTACTGGAGAACCAACTACTCTAAGCAGTGAGACACATATACCCGAGGAAAAAAGTTTGCTAAATCTTAGCACAAATGCAGATTCCCAGAGCTCTTCTGATTTTGAAGTTCCCTCAACTCCAGAAGCTGAGTTACCTAAACGAGAGCATTTACAATATTTATATGAGAAGCTGGCAACTGGTGAGAGTATAGCAGTCAAAAAAAGAAAATGCTCACTCTTAGATACCGCAGAGAACCTCTACTTCCAATCGCACCATCATCACCATCACCATCACCACCATGATTACAAGGATGACGACGATAAGTGA
Expression and purification of full-length DCLRE1C (aa 1-692)
The baculovirus generation and expression of the full length DCLRE1C was perfomed in a similar manner as the catalytic domain. Instead of infection with 1.5 mL of P2 Virus, 3.0 mL of P2 virus was used to infect SF9 cell at 2 x 106 cells/ mL for the expression of the full-length construct. Cell harvest and the initial IMAC purification step was performed as described above. Following IMAC purification TEV cleavage overnight in dialysis buffer (50 mM HEPES pH 7.5, 0.5 M NaCl, 5% glycerol and 1 mM TCEP). The protein was then passed through a 5 mL Ni-sepharose column, and the flowthrough fractions were collected. DCLRE1C protein was then concentrated on a centrifugal concentrator (Amicon®, MWCO 30 kDa) before loading on a Superdex S200 HR 16/60 gel filtration column in dialysis buffer. Fractions containing purified DCLRE1C protein were pooled and concentrated to 10 mg/mL.
Protein crystallisation and compound soaking
The catalytic domain of DCLRE1C was crystallised using a sitting drop vapour diffusion method by mixing 50 nL protein with 50 nL crystallisation solution comprising 0.2 M ammonium chloride, 20% PEG 3350 and 20 nL of crystal seed solution obtained from previous crystallisation experiments. The crystals were grown at 4 °C. Crystals grew after 1 day and reached maximum size within 1 week. Artemis protein crystal was soaked with 5 mM ceftriaxone in crystallisation solution supplemented with the addition of 20% ethylene glycol solution for one hour prior to flash freezing in liquid nitrogen. Data was collected at Diamond Light Source beamlines I03, I04 and I24, unless otherwise stated.
Structure determination
PDB Code 6TT5
Data Collection: Data were collected to 1.50 Å resolution at Diamond light source beamline I04 and was processed using DIALS.
Data Processing: The structure was solved by molecular replacement using the program PHASER and the structure of DCLRE1A (5Q2A) as a search model. Refinement was performed using REFMAC to a final Rfactor = 17 %, Rfree = 19 %.
PDB Code 7AF1
Data Collection: Data were collected to 1.70 Å resolution at Diamond light source beamline I24 and was processed using DIALS.
Data Processing: The structure was solved by molecular replacement using the program PHASER and the structure of DCLRE1C (6TT5) as a search model. Refinement was performed using REFMAC to a final Rfactor = 19 %, Rfree = 21 %.
PDB Code 7APV
Data Collection: Data were collected to 1.95 Å resolution at Diamond light source beamline I03 and was processed using DIALS.
Data Processing: The structure was solved by molecular replacement using the program PHASER and the structure of DCLRE1C (6TT5) as a search model. Refinement was performed using REFMAC to a final Rfactor = 18 %, Rfree = 23 %.
Assays
Gel-based nuclease assays
Standard nuclease assays were carried out in 10 μL reactions containing 20 mM HEPES-KOH, pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.05% Triton X-100, 5% glycerol, 0.5 mM TCEP and the indicated amount of Artemis enzyme. Reactions were started by the addition of 3’ radiolabelled (α-32P-dATP) DNA substrate (10 nM) incubated at 37 °C for the indicated time period and quenched by the addition of 10 μL stop solution (95% formamide, 10 mM EDTA, 0.25% xylene cyanol, 0.25% bromophenol blue) and incubating at 95 °C for 3 minutes.
Reactions were analysed by 20% denaturing polyacrylamide gel electrophoresis (40% solution of 19:1 acrylamide:bis-acrylamide, BioRad) and 7 M urea (Sigma Aldrich)) in 1 x TBE (Tris-borate EDTA) buffer. Electrophoresis was carried out at 700 V for 75 minutes; gels were subsequently fixed for 40 minutes in a 50% methanol, 10% acetic acid solution, and dried at 80 °C for two hours under a vacuum. Dried gels were exposed to a Kodak phosphorimager screen and scanned using a Typhoon 9500 instrument (GE).
High-throughput fluorescence-based nuclease assay.
The protocol of Lee et al [39] was adapted for structure-specific endonuclease activity. Briefly, a ssDNA substrate was utilised, containing a 5’ fluorescein-conjugated T and a 3’ BHQ-1-conjugated T (5'- [FITC] TAA TTA ATA ATA GAT CAC CT [BHQ1] - 3'). Prior to endonucleolytic incision the intact substrate does not fluoresce due to the proximity of the BHQ-1 to the fluorescein. However, following endonucleolytic incision by Artemis/DCLRE1C there is uncoupling of the fluorescein-T and the BHQ-1 and a concomitant increase in fluorescence.
The fluorescence assays were performed at 37 °C in 384-well format (black plate, clear bottom) using PheraStar plate reader by BMG Labtech. The fluorescence spectra were measured using a PHERAstar FSX (excitation: 495 nm; emission: 525 nm) with readings taken every 150 seconds. Reactions were performed in a final volume of 25 μL in buffer containing 20 mM HEPES-KOH, pH 7.5; 50 mM KCl, 10 mM MgCl2; 0.05% Triton-X100; 0.5 mM TCEP, 5% glycerol). To determine IC50, inhibitors (at increasing concentrations) were incubated with the protein for 10 minutes at room temperature, before the reaction was started with the addition of DNA substrate.
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