(A) Schematic of the D-loop reaction. (B) Representative autoradiographs showing the final invasion product for reactions in the presence of Mg²⁺, Ca²⁺, or Mn²⁺, and with or without an ATP regeneration system. (C and D) Quantitation of D-loop formation time courses. Reactions including the ATP regeneration system are indicated along the x-axis. Results are expressed as fold increase relative to WT SsoRadA with Ca²⁺ and ATP regeneration at the 1-min time point. Results shown in (D) are identical to those for WT SsoRadA data (C) in the presence of Mg²⁺, but with reduced y-axis compression to show fold change. For panels (C and D) bars with crosshatch patterning represent reactions that include the ATP regeneration system. All data are the averages of at least three reactions and error bars are ± standard deviation.