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- Identification of Potassium-Dependent and -Independent Components: MATERIALS AND METHODS(11)

Plasmid DNA Degradation Assays with Purified Nucleases

To further test for effects of potassium on nuclease activity, the pBSKII plasmid (Stratagene), linearized with the £coRI restriction enzyme (Boehringer-Mannheim), was used as a substrate for nuclease attack. The reaction buffer, consisting of 100 mM Tris-HCl (pH 7.5 for DNase-I and pH 4.6 for DNase-II), 10 mM CaCl2, and 10 mM MgCl2, was prepared without or with 0.01-0.1 U of DNase-I (Boehringer-Mannheim) or 0.01-0.1 U DNase-II (Calbi-ochem). The nucleases were preincubated for 5 min at 37°C in the absence or presence of KCl, NaCl, or LiCl, each at a final concentration of 150 mM. Sodium aurothiomalate (1 mM final) was also included as a positive control for these experiments because we have recently shown that this compound is a potent nuclease inhibitor.

Linearized plasmid was then added to the tubes (0.8 ^g/reaction, 20 ^l total reaction volume), and samples were incubated for 3 min at 4°C (DNase-I) or 37°C (DNase-II), mixed with 2 ^l of 0.5 mM EDTA and 1.3 ml 10% sodium dodecylsul-fate, and placed on ice to stop the reaction. Samples were then quickly mixed with gel-loading buffer, electrophoresed through 1% agarose gels (1 h, 100 V), stained with ethi-dium bromide, and visualized by UV-transillumination. A 1-kb DNA ladder (Gibco-BRL Life Technologies) was included for DNA size estimates.

Data Presentation and Analysis

All experiments were independently replicated at least three times. For qualitative analysis, a representative autoradiogram or photograph is presented where appropriate. Quantitative results represent the mean ± SEM of combined data from the replicate experiments. Statistical differences (P < 0.05) between mean values were analyzed by one-way analysis of variance, followed by Scheffe’s F-test.

September 30, 2013 Cells
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