Journal: Journal of Bacteriology
Article Title: Efficient Strand Transfer by the RadA Recombinase from the Hyperthermophilic Archaeon Desulfurococcus amylolyticus
Figure Lengend Snippet: DNA strand exchange reaction. A scheme of the reaction (a), the temperature (b) and kinetic (c) characteristics, and control reactions (d) are shown. In panels b and c, the reaction mixture (20 μl) contained TAcMD buffer (pH 7.9), 2.5 mM ATP, 8 μM RadA Da , 8 μM M13mp18 ssDNA, and 16 μM linearized M13mp18 dsDNA. The latter was added to initiate the reaction after 5 min of preincubation at the temperature indicated. In panel d (homologous controls, lanes 1 to 5), the reaction mixture (20 μl) was the same as described above, but either without RadA Da (lane 1) or with RadA Da degraded by proteinase K (PrK) (lane 2); a complete mixture (lane 3); the same mixture as in lane 3, but without ATP (lane 4); or the same mixture as in lane 3, but without Mg +2 (lane 5). In panel d (heterologous controls, lanes 6 to 8), the reaction mixture (20 μl) contained TAcMD buffer (pH 7.9), 2.5 mM ATP, 8 μM RadA Da , 8 μM M13mp18 ssDNA, and 16 μM linearized φX174 dsDNA (lane 6) or 8 μM φX174 ssDNA and 16 μM linearized M13mp18 dsDNA (lane 7), or 8 μM φX174 ssDNA and 16 μM linearized φX174 dsDNA (lane 8, showing the strand exchange reaction with φX174 DNA substrates).
Article Snippet: Bacteriophage φX174 DNA (RFI and circular ssDNA) was obtained from New England Biolabs.