This protocol details the BER synthesis assay using wild-type and PARP-deficient mouse embryonic fibroblast extracts and a plasmid containing a single abasic site at a defined location which allows fine mapping of the repair pathways. This assay is routinely used to investigate BER using mammalian cell extracts and is adapted from Frosina et al., 1996.
I. Construction of DNA plasmids containing a single abasic site
Materials :
PGEM-3Zf(+) single-stranded DNA prepared from the phagemid pGEM-3Zf according to the manufacturer’s instructions (Promega)
Oligonucleotides :
5’-GATCCTCTAGAGUCGACCTGCA-3’ (contains an uracil, U)
5’- GATCCTCTAGA8oxoGTCGACCTGCA-3’ (contains an 8-oxoguanine, 8-oxoG)
5’-GATCCTCTAGAGTCGACCTGCA-3’ (control)
Enzymes :
T4 polynucleotide kinase (Biolabs)
T4 DNA polymerase, T4 gene 32 protein and T4 DNA ligase (Boehringer Mannheim)
E.coli uracil DNA glycosylase and S. cerevisiae 8-oxoguanine DNA glycosylase (kindly provided by S. Boiteux, CEA, Fontenay-aux Roses, France)
Polynucleotide kinase buffer (5x) : 500 mM Tris-HCl (pH 7.4), 50 mM MgCl2, 500 mM DTT, 4 mM ATP
Sephadex G-50 column prepared in TE (pH 8.0) according to Molecular Cloning Protocols (Sambrook Fritsch Maniatis)
Annealing buffer (x10) : 200 mM Tris-HCl (pH 7.4), 20 mM MgCl2, 500 mM NaCl
Synthesis buffer (x10) : 175 mM Tris-HCl (pH 7.4), 37.5 mM MgCl2, 215 mM DTT, 7.5 mM ATP, 0.4 mM each dATP, dTTP, dCTP, dGTP
TE (pH 8.0) : 10 mM Tris-Hcl pH 8.0, 1 mM EDTA
Procedure :
Phosphorylation of oligonucleotides
1. Add 2 mg of oligonucleotide to a 1.5-ml microcentrifuge tube
2. Add 6 ml of 5x polynucleotide kinase buffer and bring the total volume to 29 ml with Milli-Q water
3. Add 10 units of T4 polynucleotide kinase, mix and incubate at 37 °C for 45 min
4. Bring up the temperature to 65 °C for 10 min to denaturate the enzyme
5. Add 20 ml TE buffer to bring the total volume to 50 ml
6. Load the sample on G-50 Sephadex column and spin for 4 min at 1500 rpm at 20 °C
7. Collect the flowthrough in a microcentrifuge tube
8. Store the phosphorylated oligonucleotide frozen at –20°C until further use.
Annealing of phosphorylated oligonucleotides to single-stranded pGEM-3Zf(+)
1. Add 10 ml of ss pGEM-3Zf(+) to a 1.5-ml microcentrifuge tube
2. Add 3 ml of 10x annealing buffer and 2 ml of Milli-Q water
3. Add 15 ml of phosphorylated oligonucleotide, mix and incubate at 75 °C for 5 min, then allow it to cool from 75°C to 30°C over 30 min
Synthesis of double-stranded pGEM-3Zf containing a single uracil (pGEM-U) or 8-oxoguanine (pGEM-8oxoG) lesions
1. Add 30 ml of annealed DNA to a 1.5-ml microcentrifuge tube
2. Add 3.6 ml of 10x synthesis buffer
3. Sequentially add 2.5 mg T4 gene 32 protein, 1 unit of T4 DNA ligase and 1 unit of T4 DNA polymerase, mix and incubate on ice for 5 min at room temperature for the next 5 min and finally at 37 °C for 90 min
4. Add one volume of phenol-chloroforme-isoamyl alcohol (25:24:1), vortex and spin for 5 min at 13,000 rpm
5. Keep the supernatant and add one volume of chloroforme-isoamyl alcohol (24:1), vortex and spin for 3 min at 13,000 rpm
6. Keep the supernatant and precipitate DNA with 0.1 volume of 3 M sodium acetate and 2 volumes of ethanol, overnight at -80°C
7. Spin for 30 min at 13,000 rpm at 4°C, dry the DNA pellet and resuspend in TE buffer
8. Double-stranded plasmids are then purified by cesium chloride equilibrium centrifugation according to standard Molecular Cloning Protocols (Sambrook, Fritsch, Maniatis)
Synthesis of double-stranded pGEM-3Zf containing a single abasic site (pGEM-AP)
Abasic sites can be produced by either monofunctional DNA glycosylases (ex : uracil-DNA glycosylase, UDG) that remove the altered base without cleaving the phosphodiester bond adjacent to the lesion or bifunctional DNA glycosylases (ex : 8-oxoguanine DNA glycosylase, OGG1) that cleave the DNA strand 3’ of the resulting abasic site by a b-elimination reaction. Both natural and incised abasic sites are repaired in a different way by the BER machinery but in a PARP-1-dependent reaction (Dantzer et al., 2000 ; Frosina et al., 1996).
Incubate the plasmid molecule pGEM-U with E.coli uracil DNA glycosylase and the plasmid pGEM-8-oxoG with S. cerevisiae 8-oxoguanine DNA glycosylase in the appropriate reaction buffers according to van der Kemp et al., 1996).
II. Whole cell extracts of wild-type and PARP-1 deficient 3T3 cells
Materials :
Ice cold PBS 1x
500 mM phenylmethylsulfonylfluoride (PMSF) dissolved in acetone. Store at –20°C.
Protease inhibitor cocktail tablets : Complete Mini EDTA-free (Roche)
Hypotonic lysis buffer (pH 7.9) : 10 mM Tris-HCl, 1 mM EDTA, 5 mM DTT, 0.5 mM spermidine, 0.1 mM spermine. Keep at 4°C.
Sucrose-glycerol buffer (pH 7.9) : 50 mM Tris-HCl, 10 mM MgCl2, 2 mM DTT, 25% sucrose, 50% glycerol. Keep at 4°C.
Dialysis buffer (pH 7.9) : 25 mM Hepes-KOH, 100 mM KCl, 12 mM MgCl2, 1 mM EDTA, 17% glycerol, 2 mM DTT ; adjust to pH 7.9 with 5 M KOH. Keep at 4°C.
Solid ammonium sulfate
Saturated ammonium sulfate solution neutralized to pH 7.0 with NaOH
Procedure :
1. Grow cells up to 1 x 109 cells
2. Wash cells twice with ice-cold PBS, scrape the cells in PBS and pellet at 1300 rpm for 5 min
3. Carefully remove the supernatant and measure the packed cell volume (PCV)
4. Resuspend the cells in 4 x PCV of ice cold hypotonic lysis buffer and add 5 ml PMSF and 1 tablet protease inhibitor cocktail per ml PCV
5. Leave the cells to swell for 20 min on ice
6. Pour the cell suspension in a glass homogenizer with a Teflon pestle and homogenize on ice with 20 strokes
7. All following steps are performed in the cold room. Pour the homogenate in a glass beaker containing a magnetic stirrer and stir very slowly
8. Add dropwise 4 x PCV of sucrose/glycerol buffer under continuous stirring
9. Add dropwise 1 x PCV of saturated ammonium sulfate and stir for 30 min
10. Pour the viscous suspension to appropriate tubes and centrifuge for 3 hours at 42,000 rpm using a SW55 rotor at 4°C
11. Remove the supernatant leaving the last 1 ml in the tube and measure the volume (usually 6-7 ml for 1 ml PCV)
12. Pour the supernatant in a glass beaker containing a magnetic stirrer, stirr slowly and add 0.33 g of pure ammonium sulfate per ml of solution
13. When dissolved, add 10 ml of 1M NaOH per g of ammonium sulfate added. Continue stirring for 30 min
14. Pour the solution in an appropriate tube and centrifuge for 20 min at 11,000g at 4°C
15. Carefully remove the supernatant with a pipette, leaving the pellet as dry as possible
16. Carefully resuspend the pellet just by mixing with a 1 ml Gilson tip in dialysis buffer (0.05 volume of the high speed supernatant)
17. Dialyze for 2 hours against 500 ml of dialysis buffer. Change the buffer and dialyze again for additional 12 hours
18. Centrifuge the dialyzed extract for 10 min at 15,800g to remove the precipitate and transfer the whole cell extract to a new tube
19. Dispense 50 ml aliquots into cryotubes, freeze on dry ice and store at –80°C
III. Repair assay
pGEM-AP or pGEM-control plasmids are incubated for 3 hours with cell-free extracts of wild-type and PARP-1 deficient 3T3 cells under standard repair conditions. To measure the overall repair (short-patch repair (SPR) + long-patch repair (LPR)), repair replication is performed in the presence of alpha-[32P]-dTTP and plasmid DNA are digested with SmaI and HindIII to release a 33-bp fragment. To measure the long-patch repair efficiency (LPR), repair replication is performed in the presence of alpha-[32P]-dCTP and plasmid DNA are digested with HincII and HindIII to release a 16-bp fragment downstream to the lesion.
Materials :
Synthesis buffer (5x) : 225 mM Hepes/KOH (pH 7.8), 300 mM KCl, 37.5 mM MgCl2, 4.5 mM DTT, 50 mM each dNTP, 10 mM ATP, 200 mM phosphocreatine, 90 mg BSA. Store in aliquots at –80°C
Creatine phosphokinase (CPK) (Type 1, Sigma), 2.5 mg/ml dissolved in 5 mM glycine (pH 9.0), 50% glycerol. Store in aliquots at –80°C
EDTA 0.5 M
RNase A : 2 mg/ml dissolved in H2O. Store in aliquots at –20°C
SDS 10%
Proteinase K, 2 mg/ml dissolved in H2O. Store in aliquots at –20°C
Denaturating loading buffer : 80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue
alpha-[32P]-labeled dNTP solution (alpha-[32P]-dTTP or alpha-[32P]-dCTP, 3000 Ci/mmol, 10 mCi/ml, Amersham)
Procedure :
Repair assay
1. Add 400 ng of plasmid construct to a 1.5-ml microcentrifuge tube
2. Add 50 mg of mouse whole cell extract
3. Add 10 ml of 5x synthesis buffer and 1 ml of creatine phosphokinase
4. Bring the total volume to 49 ml with Milli-Q water
5. Add 1 ml of [32P]-labeled dNTP solution (alpha-[32P]-dTTP or alpha-[32P]-dCTP), mix and incubate at 30°C for 3 hours
6. Stop the reaction by adding 2 ml of EDTA 0.5 M and keep on ice
7. Add 2 ml of RNase 2 mg/ml and incubate at 37 °C for 10 min
8. Add 3 ml SDS 10% and 6 ml proteinase K 2 mg/ml and incubate at 37°C for 30 min
Purification of the DNA product
1. Add an equal volume of phenol, vortex and centrifuge for 5 min at 14,000 rpm at room temperature
2. Transfer the supernatant to a new tube
3. Extract the phenol phase again with an equal volume of TE, vortex and centrifuge as in step 1
4. Pool supernatants from step 1 and 2, add half volume of phenol, vortex and centrifuge as in step 1
5. Add an equal volume of chloroform/isoamyl alcohol (24:1), vortex and centrifuge as in step 1
6. To the supernatant containing the plasmid, add 1/4 volume of 7.5 M ammonium acetate and 2.5 volumes of cold ethanol ; mix and keep overnight at –80°C
7. Centrifuge for 30 min at 14,000 rpm at 4°C, remove carefully the supernatant and air-dry the pellet
8. Resuspend the pellet in 15 ml TE and proceed to digestion by restriction enzymes
Digestion with appropriate restriction enzymes
1. Incubate the DNA with the appropriate restriction enzymes according to the manufacturer’s instructions
2. Extract and precipitate the DNA as described above
3. Resuspend the precipitate in 6 ml denaturating loading buffer
4. Heat the samples at 95°C for 2 min
5. Load onto a 15% polyacrylamide gel containing 7 M urea in 90 mM-Tris-borate/2 mM EDTA (pH 8.8)
6. Electrophorese the samples at 60 mA for 2 hours
7. Dry the gel and expose it to autoradiography
References :
Dantzer, F., de La Rubia, G. Ménissier-de Murcia, J., Hostomsky, Z., de Murcia, G., Schreiber, V. (2000) Base excision repair is impaired in mammalian cells lacking poly(ADP- ribose) polymerase-1. Biochemistry 39, 7559-69.
Frosina G., Fortini P., Rossi O., Carrozzino F., Raspaglio G., Cox L.S., Lane D.P., Abbondandolo A., Dogliotti E. (1996) Two pathways for base excision repair in mammalian cells. J. Biol. Chem. 271, 9573-9578.
van der Kemp, P.A., Thomas, D., Barbey, R., de Oliveira, R., and Boiteux, S. (1996) Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine. Proc. Natl. Acad. Sci. USA 93, 5197-202.
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