Radiation Effects on BamHI Endonuclease Activity

Pre- and postirradiation activity of BamHI was determined as described previously (15 (link), 20 (link)) with some modifications. BamHI (5,000 U/μL) (without bovine serum albumin [BSA]) (New England Biolabs, Ipswich, MA, USA) was diluted in D. radiodurans or in C. elegans ultrafiltrate to 0.54 U/μL. Then, 200 μL of the BamHI mixture was gamma-irradiated aerobically on ice. Following irradiation, 20 μL of each radiation-treated BamHI sample was assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 μL) containing 125 ng lambda phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl₂, and 1 mM dithiothreitol (New England Biolabs; buffer 3). BamHI/lambda DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis.