Figure 3.

Effects of amino-acid replacements for Asp-161 in loop L1 on the double-stranded DNA-dependent ATPase activity of RecA at various MgCl₂ concentrations. [¹⁴C or α-³²P]ATP (26 nmol at 1.3 mM) was incubated with 2.0-μM mutant recA or RecA-wt in the presence of the indicated amounts of MgCl₂ and 10-μM pGSat4 (in (A)) or 23-μM pBluescript SK(-) (in (B)) supercoiled double-stranded DNA (form I DNA). The reaction products were fractionated by thin layer chromatography, and the amounts of ATP hydrolyzed were determined by analyzing the ¹⁴C- or ³²P-signals. (A) Effects of the various amino-acid replacements of Asp-161, and the Ala-replacement of Ser-162. The double-stranded DNA was the negatively supercoiled closed circular form (form I), and ¹⁴C-labeled ATP was used. Amino-acid replacements of mutant recAs used in these experiments are indicated within each panel. Each point indicates the average obtained from two to three independent experiments. (B) Effects of variations in the form of the double-stranded DNA. The double-stranded DNA used was form I (black closed symbols), the nicked circular form (grey open symbols) or the linear form (black open symbols). [α-³²P]ATP was used in these experiments. ▼, ▽ (inverted triangles), recA-D161A; ●, ○ (circular symbols), RecA-wt. In (B) and the following figures, each point indicates the average obtained from more than three independent experiments. Some symbols are larger than the error bars, and thus cover them.