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Deoxyguanosine triphosphate
Deoxyguanosine triphosphate
Deoxyguanosine triphosphte is a nucleoside triphosphate essential for DNA synthesis and repair.
It serves as the building block for the genetic material, providing the guanine base and deoxyribose sugar needed to construct DNA strands.
Optimizing deoxyguanosine triphosphate research is critical for advancing understaning of fundamental cellular processes and developing therapies that target DNA-related pathologies.
PubCompare.ai's AI-driven protocols can help researchers discover the best methods from literature, preprints, and patents, improving reproducibility and accuracy of deoxyguanosine triphospahte studies.
It serves as the building block for the genetic material, providing the guanine base and deoxyribose sugar needed to construct DNA strands.
Optimizing deoxyguanosine triphosphate research is critical for advancing understaning of fundamental cellular processes and developing therapies that target DNA-related pathologies.
PubCompare.ai's AI-driven protocols can help researchers discover the best methods from literature, preprints, and patents, improving reproducibility and accuracy of deoxyguanosine triphospahte studies.
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Cell lines were cultured in DMEM supplemented with 10% FCS. BS-Ctrl(BLM), BS-BLM cells, BS-Ctrl(CDA) and BS-CDA were obtained and cultured as previously described [25 (link)].
The BS-BLM-CDA cell line was obtained by transfecting BS-BLM cells with a vector containing the full-length CDA cDNA (NM001785), with JetPEI reagent. After 48 h, selection was carried out with 0.2 μg.ml− 1 puromycin (Invivogen) and 500 μg.ml− 1 G418 (Euromedex). Individual colonies were isolated and maintained in culture with 0.1 μg.ml− 1 puromycin and 500 μg.ml− 1 G418.
HeLa-Ctrl(CDA) and HeLa-shCDA cells were obtained by transfecting cells with an empty pGIPZ vector or with the same vector encoding a short hairpin RNA sequence directed against CDA (Open Biosystems, clone V3LHS_369299), respectively, with JetPEI reagent. After 48 h, transfectants were selected on 1–5 μg.ml− 1 puromycin (Invivogen). Individual colonies were isolated and cultured in medium containing 1 μg.ml− 1 puromycin.
HeLa-Ctrl(PARP-1) and HeLa-shPARP-1 cells were cultured as previously described [46 (link)].
For siRNA transfection assays, 3×105 HeLa cells or 8×105 BS-Ctrl(BLM), BS-BLM, BS-Ctrl(CDA), BS-CDA, or BS-BLM-CDA cells were used to seed the wells of a six-well plate. Cells were transfected with an siRNA specific for BLM or CDA (ON-TARGETplus SMARTpool, Dharmacon) or negative control siRNAs (ON-TARGETplus siCONTROL Non Targeting Pool, Dharmacon; 100 nM final concentration) for 48 h for BLM, or twice successively, for a total of 120 h for CDA, in the presence of DharmaFECT 1 (Dharmacon).
Deoxyuridine (dU), deoxycytidine (dC), deoxyuridine triphosphate (dUTP), deoxycytidine triphosphate (dCTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (dTTP) were provided by Sigma Aldrich (D5412; D0779; D4001, D4635, D6500, D4010 and T0251 respectively); tetrahydrouridine (THU) was provided by Calbiochem (584222); camptothecin (CPT) was provided by Sigma Aldrich (C9911) and olaparib was provided by SelleckChem (S1060). THU and dU were added to the cell culture medium at a final concentration of 100 μM, for 96 h (2x48 h). Other drugs were added to the cell culture medium at the following concentrations: dC, 1 mM; H2O2, 30 μM; camptothecin, 2 pM; olaparib, 1 μM.
All cells were routinely checked for mycoplasma infection.
The BS-BLM-CDA cell line was obtained by transfecting BS-BLM cells with a vector containing the full-length CDA cDNA (NM001785), with JetPEI reagent. After 48 h, selection was carried out with 0.2 μg.ml− 1 puromycin (Invivogen) and 500 μg.ml− 1 G418 (Euromedex). Individual colonies were isolated and maintained in culture with 0.1 μg.ml− 1 puromycin and 500 μg.ml− 1 G418.
HeLa-Ctrl(CDA) and HeLa-shCDA cells were obtained by transfecting cells with an empty pGIPZ vector or with the same vector encoding a short hairpin RNA sequence directed against CDA (Open Biosystems, clone V3LHS_369299), respectively, with JetPEI reagent. After 48 h, transfectants were selected on 1–5 μg.ml− 1 puromycin (Invivogen). Individual colonies were isolated and cultured in medium containing 1 μg.ml− 1 puromycin.
HeLa-Ctrl(PARP-1) and HeLa-shPARP-1 cells were cultured as previously described [46 (link)].
For siRNA transfection assays, 3×105 HeLa cells or 8×105 BS-Ctrl(BLM), BS-BLM, BS-Ctrl(CDA), BS-CDA, or BS-BLM-CDA cells were used to seed the wells of a six-well plate. Cells were transfected with an siRNA specific for BLM or CDA (ON-TARGETplus SMARTpool, Dharmacon) or negative control siRNAs (ON-TARGETplus siCONTROL Non Targeting Pool, Dharmacon; 100 nM final concentration) for 48 h for BLM, or twice successively, for a total of 120 h for CDA, in the presence of DharmaFECT 1 (Dharmacon).
Deoxyuridine (dU), deoxycytidine (dC), deoxyuridine triphosphate (dUTP), deoxycytidine triphosphate (dCTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (dTTP) were provided by Sigma Aldrich (D5412; D0779; D4001, D4635, D6500, D4010 and T0251 respectively); tetrahydrouridine (THU) was provided by Calbiochem (584222); camptothecin (CPT) was provided by Sigma Aldrich (C9911) and olaparib was provided by SelleckChem (S1060). THU and dU were added to the cell culture medium at a final concentration of 100 μM, for 96 h (2x48 h). Other drugs were added to the cell culture medium at the following concentrations: dC, 1 mM; H2O2, 30 μM; camptothecin, 2 pM; olaparib, 1 μM.
All cells were routinely checked for mycoplasma infection.
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To perform error prone PCR, we combined 100 ng of the template plasmid (Aga2p-HA-LOV(-2)-TEVcs(high affinity)-FLAG in pCTCON2) with 0.4 μM forward and reverse primers that anneal to the sequences just outside the 5′ and 3′ ends of the AsLOV2 gene (sequences below), 2 mM MgCl2, 5 units of Taq polymerase (NEB), and 2 μM each of the mutagenic nucleotide analogs 8-oxo-2′-deoxyguanosine-5′triphosphate (8-oxo-dGTP) and 2′-deoxy-p-nucleoside-5′-triphosphate (dPTP) in a 50 μL volume (2 reactions were required).
The PCR was run for 20 cycles with annealing temperature of 58 °C in each cycle. Then the PCR product was gel-purified, and re-amplified for another 30 cycles with 0.4 μM forward and reverse primers that introduce ~45 bp of overlap with both ends of the vector:
PCR annealing temperature was 58 °C and regular dNTPs (10 μM) and 5 units of Taq polymerase in 50 μL volume was used. The PCR products from four separate reactions were combined purified by gel extraction.
Separately, we linearized the Aga2p-HA-LOV(-2)-TEVcs(high)-FLAG-pCTCON2 plasmid by digesting with BamHI and NheI restriction enzymes for 3 hrs at 37°C. These enzymes cut the gene just upstream of the LOV domain and downstream of the TEVcs. The linearized plasmid was purified by gel extraction. We then combined 1 μg of linearized vector with 4 ug of mutagenized LOV PCR product from above, concentrated using pellet paint (Millipore) according to the manufacturer’s protocols (precipitation of DNA with ethanol and sodium acetate, and resuspended in 10 μL ddH2O.
In parallel, fresh electrocompetent EBY100 cells were prepared. EBY100 cells are passaged at least two times before this procedure to ensure that the cells are healthy. We used a 2–3 mL saturated culture of EBY100 cells to inoculate 100 mL of fresh YPD media (no older than 2 months), and grew the cells with shaking at 2000 rpm at 30 °C for 6–8 hrs until the OD600 reached 1.5–1.8. The cells were then harvested by centrifugation for three minutes at 3000 rpm and resuspended in 50 mL of sterile 100 mM lithium acetate in water, by vigorous shaking. Fresh sterile DTT (1 M stock solution, made on the same day) was added to the yeast cells to a final concentration of 10 mM. The cells were incubated with shaking at 220 rpm for 12 minutes at 30 °C (necessary to ensure adequate oxygenation). Then cells were pelleted at 4 °C by centrifugation at 3000 rpm for 3 minutes and washed once with 25 mL ice-cold sterile water, pelleted again, and resuspended in 1 mL ice cold sterile water.
The concentrated mixed DNA from above was combined with 250 μL of electrocompetent EBY100 cells on ice and then electroporated using a Bio-Rad Gene pulser XCell. The electroporated cells were immediately rescued with 2 mL pre-warmed YPD media and then incubated at 30 °C for 1 hr without shaking. 10 uL of this solution was used to determine transformation efficiency, and the remainder was spun down to remove the YDP media and resuspended in 100 mL SDCAA media supplemented with 50 units/mL penicillin and 50 μg/mL streptomycin. The culture was grown at 30 °C with shaking at 220 rpm for 1 day, before induction of protein expression and positive selection as described below (“Yeast display selection”).
Transformation efficiency of our LOV library into EBY100 yeast was 3.6 × 107. DNA sequencing of 12 individual clones (Figure SI-3 ) showed that each clone had 0–2 nucleotides changed relative to original AsLOV2 template.
The PCR was run for 20 cycles with annealing temperature of 58 °C in each cycle. Then the PCR product was gel-purified, and re-amplified for another 30 cycles with 0.4 μM forward and reverse primers that introduce ~45 bp of overlap with both ends of the vector:
PCR annealing temperature was 58 °C and regular dNTPs (10 μM) and 5 units of Taq polymerase in 50 μL volume was used. The PCR products from four separate reactions were combined purified by gel extraction.
Separately, we linearized the Aga2p-HA-LOV(-2)-TEVcs(high)-FLAG-pCTCON2 plasmid by digesting with BamHI and NheI restriction enzymes for 3 hrs at 37°C. These enzymes cut the gene just upstream of the LOV domain and downstream of the TEVcs. The linearized plasmid was purified by gel extraction. We then combined 1 μg of linearized vector with 4 ug of mutagenized LOV PCR product from above, concentrated using pellet paint (Millipore) according to the manufacturer’s protocols (precipitation of DNA with ethanol and sodium acetate, and resuspended in 10 μL ddH2O.
In parallel, fresh electrocompetent EBY100 cells were prepared. EBY100 cells are passaged at least two times before this procedure to ensure that the cells are healthy. We used a 2–3 mL saturated culture of EBY100 cells to inoculate 100 mL of fresh YPD media (no older than 2 months), and grew the cells with shaking at 2000 rpm at 30 °C for 6–8 hrs until the OD600 reached 1.5–1.8. The cells were then harvested by centrifugation for three minutes at 3000 rpm and resuspended in 50 mL of sterile 100 mM lithium acetate in water, by vigorous shaking. Fresh sterile DTT (1 M stock solution, made on the same day) was added to the yeast cells to a final concentration of 10 mM. The cells were incubated with shaking at 220 rpm for 12 minutes at 30 °C (necessary to ensure adequate oxygenation). Then cells were pelleted at 4 °C by centrifugation at 3000 rpm for 3 minutes and washed once with 25 mL ice-cold sterile water, pelleted again, and resuspended in 1 mL ice cold sterile water.
The concentrated mixed DNA from above was combined with 250 μL of electrocompetent EBY100 cells on ice and then electroporated using a Bio-Rad Gene pulser XCell. The electroporated cells were immediately rescued with 2 mL pre-warmed YPD media and then incubated at 30 °C for 1 hr without shaking. 10 uL of this solution was used to determine transformation efficiency, and the remainder was spun down to remove the YDP media and resuspended in 100 mL SDCAA media supplemented with 50 units/mL penicillin and 50 μg/mL streptomycin. The culture was grown at 30 °C with shaking at 220 rpm for 1 day, before induction of protein expression and positive selection as described below (“Yeast display selection”).
Transformation efficiency of our LOV library into EBY100 yeast was 3.6 × 107. DNA sequencing of 12 individual clones (
Most recents protocols related to «Deoxyguanosine triphosphate»
All PER experiments were incubated at 37°C for the indicated times, usually with 1× ThermoPol buffer with supplemented magnesium [20 mM tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 12 mM MgSO4, and 0.1% Triton X-100] and 0.8 U/μl of Bst. Large fragment polymerase (purchased from New England Biolabs, M0275L) and 1 μM of the appropriate dHTPs (Deoxynucleotide triphosphate solution without deoxyguanosine triphosphate, purchased from Diamond). Typically, 20 μl of reactions were quenched by heat inactivation of the enzyme at 85°C for 20 min. See tables S2 and S3 for the reaction details of each experiment.
We prepared the sequencing libraries following the manufacturer’s recommendations of Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme Biotech Co., Ltd., Nanjing, China, N406) and VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd., Nanjing, China, NR605). The details of library construction are as follows. First, we removed ribosome RNA from 200 ng total RNA using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme, N406). Then, we fragmented the RNA into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H, deoxyuridine triphosphate (dUTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP). Then, a single ‘A’ base and the adapters were subsequently added to these cDNA fragments. In order to select the appropriate cDNA fragment size for sequencing, we selected the library fragments with VAHTSTM DNA Clean Beads (Vazyme, N411). The polymerase chain reaction (PCR) amplification was performed, and the aimed products were finally purified. After cluster generation, the libraries were sequenced on an Illumina novaseq 6000 platform, and the raw fastq files of 150-bp paired-end reads were generated.
Kyoto Green was synthesized following the procedure described in Ojida et al.23 (link). Magnesium chloride (MgCl2), zinc nitrate (Zn(NO3)2), HEPES minimum 99.5% titration (C8H18N2O4S), p-nitrophynyl thymidine 5′-monophosphate (pNph-5′-TMP), sodium pyrophosphate decahydrate (NaPPi), adenosine 5′-triphosphate disodium salt (ATP), and adenosine 5′-monophosphate disodium salt (AMP) were products of Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany). Cordycepin, uridine, cytidine, inosine, thymidine, kaempferol, 2′-deoxyadenosine, 2′-deoxyinosine, 2′-deoxyuridine, and 2′-deoxyguanosine were purchased from Fujifilm Wako Pure Chemical Corporation (Japan). Myricetin, quercetin dihydrate and guanosine were commercially available from Alfa Aesar (UK). Recombinant human ENPP1 was purchased from R&D systems (Minneapolis, USA).
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Site-specific urea lesion-containing
12-mer template oligodeoxynucleotides corresponding to each urea-containing
strand were annealed with an equimolar amount of 8-mer oligodeoxynucleotide
primers (Chart 2 ) in
100 mM NaCl, 50 μM EDTA (sodium salt), and 20 mM Tris–HCl
(pH 8.5) at room temperature. They were kept on ice for 30 min. To
first make the binary complex of hPol η and urea-containing
DNA template primer, protein in 450 mM KCl, 5% glycerol, 1 mM TCEP,
3 mM DTT, and 20 mM Tris–HCl (pH 7.5), and DNA were mixed in
a 1:1.1 molar ratio, followed by incubation at room temperature for
10 min. To reduce the salt concentration to 150 mM KCl, dilution buffer
containing a concentration of 5 mM MgCl, 5% glycerol, 1 mM TCEP, 3
mM DTT, and 20 mM Tris–HCl (pH 7.5) was added. The samples
were concentrated using 10 kDa cutoff amicon centrifugal filters (Millipore
Sigma, Burlington, MA) to yield 2 mg/mL protein concentration. To
assemble ternary complexes, dNTP analogs 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dAMPnPP), 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dCMPnPP), 2′-deoxyguanosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dGMPnPP), and 2′-deoxythymidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dTMPnPP) (Jena BioScience, Jena, Germany) were added
separately to a 10 mM final concentration into a tube that contained
concentrated hPol η-DNA binary complex. Solutions were kept
on ice for 30 min before setting up 24-well crystallization plates.
For each ternary complex crystallization, we used the hanging drop
vapor diffusion method in combination with a 700 mL reservoir containing
polyethylene glycol monomethyl ether 2000 (PEG MME2000) (Hampton Research,
Aliso Viejo, CA) (14–24%), 5 mM MgCl2, and 0.1 M
MES hydrate (Millipore Sigma, Burlington, MA) at one of three pH values
of pH 5.6, pH 6.0, and pH 6.5.18 (link),30 (link) For each drop, 0.8
μL of ternary complex was mixed with 0.8 μL of a reservoir
solution. Plates were incubated either at 18 °C or at room temperature.
Crystals were observed for samples corresponding to both urea-containing
DNA strands and all four dNTPs at different pH and PEG concentrations
in 1 to 2 days. Diffraction-quality crystals were obtained after incubating
plates for 1 week.
12-mer template oligodeoxynucleotides corresponding to each urea-containing
strand were annealed with an equimolar amount of 8-mer oligodeoxynucleotide
primers (
100 mM NaCl, 50 μM EDTA (sodium salt), and 20 mM Tris–HCl
(pH 8.5) at room temperature. They were kept on ice for 30 min. To
first make the binary complex of hPol η and urea-containing
DNA template primer, protein in 450 mM KCl, 5% glycerol, 1 mM TCEP,
3 mM DTT, and 20 mM Tris–HCl (pH 7.5), and DNA were mixed in
a 1:1.1 molar ratio, followed by incubation at room temperature for
10 min. To reduce the salt concentration to 150 mM KCl, dilution buffer
containing a concentration of 5 mM MgCl, 5% glycerol, 1 mM TCEP, 3
mM DTT, and 20 mM Tris–HCl (pH 7.5) was added. The samples
were concentrated using 10 kDa cutoff amicon centrifugal filters (Millipore
Sigma, Burlington, MA) to yield 2 mg/mL protein concentration. To
assemble ternary complexes, dNTP analogs 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dAMPnPP), 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dCMPnPP), 2′-deoxyguanosine-5′-[(α,β)-imido]triphosphate
(sodium salt; dGMPnPP), and 2′-deoxythymidine-5′-[(α,β)-imido]triphosphate
(sodium salt; dTMPnPP) (Jena BioScience, Jena, Germany) were added
separately to a 10 mM final concentration into a tube that contained
concentrated hPol η-DNA binary complex. Solutions were kept
on ice for 30 min before setting up 24-well crystallization plates.
For each ternary complex crystallization, we used the hanging drop
vapor diffusion method in combination with a 700 mL reservoir containing
polyethylene glycol monomethyl ether 2000 (PEG MME2000) (Hampton Research,
Aliso Viejo, CA) (14–24%), 5 mM MgCl2, and 0.1 M
MES hydrate (Millipore Sigma, Burlington, MA) at one of three pH values
of pH 5.6, pH 6.0, and pH 6.5.18 (link),30 (link) For each drop, 0.8
μL of ternary complex was mixed with 0.8 μL of a reservoir
solution. Plates were incubated either at 18 °C or at room temperature.
Crystals were observed for samples corresponding to both urea-containing
DNA strands and all four dNTPs at different pH and PEG concentrations
in 1 to 2 days. Diffraction-quality crystals were obtained after incubating
plates for 1 week.
For our long-term experimental evolution of VIM-2, we started from the wild-type VIM-2-coding gene cloned into a low-copy number in-house plasmid with a constitutive, low expression TEM promoter and chloramphenicol resistance, which we named pIDR29 (link). We generated randomly mutagenized libraries of wild-type (WT) VIM-2 via error-prone PCR (epPCR) by adding the nucleotide analogues 8-oxo-2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP) or 2’-deoxy-P-nucleoside-5’-triphosphate (dPTP) (TriLink). Each of the two 25 μL PCR reactions consisted of 1 x GoTaq Buffer (Promega), 3 μM MgCl2, 0.1 μM of each primer, 0.2 mM of dNTPs, 1.00 U of GoTaq DNA polymerase (Promega), 1 ng of template plasmid, and either 100 μM of 8-oxo-dGTP or 1 μM of dPTP. We programmed the first PCR (error-prone PCR) as follows: an initial denaturation step (95 °C for 2 min), followed by 20 cycles of 95 °C for 30 s, 58 °C for 60 s, 72 °C for 60 s, before a final extension step (72 °C for 3 min). We subsequently purified the PCR products with the EZ.N.A.® Cycle Pure PCR Purification Kit (OMEGA Bio-tek Inc), quantified them using a NanoDrop spectrophotometer, and used them in equal parts in a subsequent PCR reaction to ensure a balance between transition versus transversion nucleotide mutations, and a specific mutation rate and increased product yield for downstream processing. The second PCR reaction used the following reagents: 5 µL of 1 ng/ µL dPTP-epPCR product, 5 µL of 1 ng/ µL 8-oxo-dGTP-epPCR product, 1 x GoTaq Buffer (Promega), 3 μM MgCl2, 0.1 μM of each primer, 0.25 mM of dNTPs, 1.0 U of GoTaq DNA polymerase (Promega) in a final volume of 50 μL. We performed the second PCR using the same program as the first, but with 35 instead of 20 amplification cycles. We then purified the PCR products with the EZNA Cycle Pure PCR Purification Kit, and digested them with NcoI (FastDigest, ThermoFisher Scientific™) and XhoI (FastDigest, ThermoFisher Scientific™) for 1 h at 37 °C. In addition, we digested the pIDR29 (link) plasmid with NcoI and XhoI, for 3 h at 37 °C. Subsequently, we purified the digested plasmid from a 1% agarose gel using gel purification columns, while we purified the digested PCR products of the mutagenized VIM-2 gene with an E.Z.N.A.® Cycle Pure PCR Purification Kit. We ligated the digested VIM-2 gene fragments with the vector using a ligation mixture (10 μL) consisting of 1 × T4 DNA ligase buffer (ThermoFisher Scientific™), 5 U of T4 DNA ligase (ThermoFisher Scientific™), 8–10 ng of prepared vector, and 30–40 ng of prepared mutagenized insert. We incubated this mixture at room temperature for 3 h. We then purified the resulting ligation products with a MicroElute kit (OMEGA Bio-tek Inc.) and eluted them with 20 μL of water.
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dGTP, Deoxyguanosine triphospahte, DNA synthesis, DNA repair, nucleoside triphosphate, guanine, deoxyribose, cellular processes, DNA-related patholigies, DNase I, 5-methyl-2′-deoxycytidine-5′-triphosphate, 5mdCTP, Phosphodiesterase I, T4 DNA ligase, Adenosine 5′-triphosphate disodium salt, In Situ Cell Death Detection Kit, Etidronate, Glutaraldehyde, Pamidronate, 3′-Deoxyguanosine-5′-Triphosphate