Scaled-down nuclease P1 for scaled-up DNA digestion.

Motivated by an interest in detecting DNA adducts in their nucleotide form by mass spectrometry (1), we sought efficient, low-cost conditions for enzymatic digestion of a relatively large amount of DNA (≥ 1 mg). Of the enzymes available for such digestion, nuclease P1, which forms deoxynucleoside-5′-monophosphates, seemed to be a good initial choice because it is widely used for digesting DNA, very stable, and relatively low in cost. However, considering our goal of detecting DNA adducts, a study of the literature revealed three uncertainties concerning the use of this enzyme for hydrolyzing a large amount of DNA. First, some workers denature the DNA first by heating (95°C–100°C) to accelerate the subsequent enzymatic digestion (2,3). Since this could both create and modify some DNA adducts, it was a condition that we wanted to avoid. Second, many variations have been reported in digestion conditions, including the amount of the enzyme, which has ranged up to a cost equivalent to about $30/mg DNA. Third, few studies have assessed the degree of digestion. Although the prior literature contains many methods for digesting DNA with nuclease P1, the one by McGall et al. (4) seemed to provide a good starting point because they digested unheated DNA at a relatively high concentration of about 0.2 mg/mL. To fully optimize nucleotide yield, we varied all of their digestion conditions. Hoechst dye binding was used to quantify the DNA before digestion, and HPLC-UV was used to quantify the nucleotides afterwards. Our recommended method is as follows: 1 mg DNA in 1.05 mL water in a plastic tube (Brinkman, Westbury, NY, USA) is treated with 200 μL 5 mM ZnCl2, 300 μL 50 mM sodium acetate, pH 5.5, and 2 U (2 μL) nuclease P1 (Roche Applied Science, Indianapolis, IN, USA, unless indicated otherwise), giving a total volume of 1.55 mL with 1.3 U/mL nuclease P1, 0.65 mM ZnCl2, and 9.7 mM sodium acetate. The tube is capped and kept for 2 h at 37°C–38°C before centrifugal ultrafiltration (2 mL Centricon YM-10; Fisher Scientific, Pittsburgh, PA, USA) and injection of 20 μL into an HPLC column to assess the yield of nucleotides. Figure 1 shows an HPLC chromatogram from such a digestion. According to our measurements, 88% of the DNA is hydrolyzed to nucleotides, but the true degree of hydrolysis may be essentially 100% considering the limited accuracy, especially of the dye-binding assay. The digestion appeared to be complete (by HPLC) after 1 h, but only about 70% hydrolysis took place after 0.5 h. Relative to the conditions reported by McGall et al. (4), mainly we increased the concentration of DNA by 3.2-fold and reduced the relative amount of enzyme by 16 times, so about $0.43 of enzyme is used per milligram of DNA. While doubling the latter amount of enzyme had no effect (tested at pH 6.0 with a 2-h digestion time in succinate buffer), half the amount of enzyme gave late-eluting peaks (at about 20 min) in the HPLC chromatogram, apparently from incomplete digestion. Our final amount of enzyme (2 U/mg DNA) is 2 times lower than the lowest value reported before (5). Most of the optimization experiments were done on 30-μg amounts of DNA. Curiously, when a relatively high dose of nuclease P1 was tested in such an experiment, deoxyadenosine (dA) formed as a side product, giving an extra peak in the HPLC chromatogram, as shown in Figure 2A. We identified this peak as dA by (i) measuring its UV spectrum, (ii) determining that it coeluted with added dA, and (iii) observing that adenosine deaminase converted this product to deoxyinosine. Note as well that the peak for dAMP is relatively larger (Figure 2B) when less nuclease P1 is added. No dA formed when DNA was omitted from the experiment, and the amount of dA did not increase with an extended incubation time. When authentic dA-5′-monophosphate was incubated with nuclease P1, the peak did not form. Nuclease P1 from Sigma-Aldrich (St. Louis, MO, USA) gave the same result. We did not characterize this event in more detail. It may be relevant that for both DNA (6) and dideoxynucleotides (7), the phosphodiester bond 3′ to an adenine nucleotide is a preferred site of hydrolysis by this enzyme. The 3′-phosphodiester linkage of some nucleobase DNA adducts resists digestion by nuclease P1, yielding dinucleotides (8) or trinucleotides (9), but this may be acceptable for our purposes. Benchmarks