Enzymatic Activity Assays for Base Excision Repair Enzymes in Cell Extracts from Vertebrate Cells

Materials and Reagents

1. Cell lines used in this study
   1. The human cell lines used are the wild-type C2ABR and AOA1 L938 (Harris et al., 2009). The AOA1 cell line was derived from the peripheral blood of a Japanese AOA1 patient and has a point mutation within the HIT domain of APTX involving substitution of proline for leucine at position 206.
   2. The DT40 cell lines are the wild-type and APTX null. The APTX null cell line has the aptx gene deletion from valine 78 onwards that inactivates APTX (Ahel et al., 2006).
   3. The mouse embryonic fibroblast cell lines are pol β^-/- and pol β^+/+ MEFs previously developed in our laboratory (Sobol et al., 1996).
2. RPMI 1640 medium with glutamine (Gibco, catalog number: 11875-093 )
   
3. DMEM high-glucose medium (HyClone, catalog number: SH30081 )
   
4. Chicken serum (Life Technologies, catalog number 16110-082 )
   
5. Glutamax-1 (Gibco, catalog number: 35050-061 )
   
6. Fetal bovine serum-FBS (HyClone, catalog number: SH30910 ).
   
7. EDTA-free protease inhibitor cocktail tablet (Roche Applied Science, catalog number: 11836170001 )
   
8. Bio-Rad Protein Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
   
9. EDTA (Sigma-Aldrich, catalog number: 93283 )
   
10. Potassium chloride-KCl (Sigma-Aldrich, catalog number: P9333 )
    
11. Sodium chloride-NaCl (Sigma-Aldrich, catalog number: S7653 )
    
12. Glycerol (Sigma-Aldrich, catalog number: G9012 )
    
13. Tissue-culture grade 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
    
14. Nonidet P 40-NP40 (Sigma-Aldrich, catalog number: 74385 )
    
15. HEPES (Sigma-Aldrich, catalog number: H3375 )
    
16. Magnesium chloride-MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
    
17. Formamide (Sigma-Aldrich, catalog number: F9037 )
    
18. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
    
19. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
    
20. Dithiothreitol-DTT (Sigma-Aldrich, catalog number: D0632 )
    
21. Sodium borohydride-NaBH₄ (Sigma-Aldrich, catalog number: 247677 )
    
22. Urea (National Diagnostic, catalog number: EC-605 )
    
23. Trizma-base (Sigma-Aldrich, catalog number: T4661 )
    
24. Boric Acid (Promega Corporation, catalog number: H5003 )
    
25. AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850 )
    
26. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678-25G )
    
27. Tetramethylethylenediamine (Sigma-Aldrich, catalog number: T9281-25ML )
    
28. Sterile water
    
29. Purified enzymes: Recombinant human DNA polymerase β [purified as described Çağlayan et al. (2014)], recombinant human APTX (Fitzgerald catalog number: 80R-1256 ), and recombinant human FEN1 [purified as described Çağlayan et al. (2014)].
    
30. DNA substrate: The gapped DNA substrate with a uracil base at position 17 at the 5'-end of the 3'-end FAM-labeled oligonucleotides. The sequence information for the upstream, downstream and template oligonucleotides were previously published Çağlayan et al. (2015).
    
31. Lysis buffer (see Recipes)
    
32. 10x reaction buffer (see Recipes)
    
33. Gel-loading buffer (see Recipes)
    
34. 10x TBE solution and 1x TBE solution as PAGE running buffer (see Recipes)
    
35. 15% Denaturing Polyacrlamide Gel or PAGE solution (see Recipes)
    

Equipment

1. Eppendorf tubes
   
2. Screw cap conical tube (15 ml)
   
3. Refrigerated table-top centrifuge
   
4. Refrigerated Eppendorf centrifuge
   
5. Table-top heat block
   
6. Tissue culture CO₂ incubators set at 34, 37, and 39.5 °C
   
7. Cell scraper
   
8. Polyacrylamide gel electrophoresis (PAGE) apparatus
   

Procedure