FIG. 5.

Endonuclease assays. (A) DNA substrates for MtuPps1 and MtuRecA. The sequences of MtuPps1 and MtuRecA homing sites (HS) inserted in pUC19 (the sequence of which appears in boldface) are underlined. (B) Cleavage assay for MtuPps1. One hundred nanograms of linearized substrate 2 was incubated with either 2.5 μg of the crude extract of MtuPps1 (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl₂ and 25 mM KCl. Substrate (S2; 2,730 bp) and products (P; 940 and 1,790 bp) were separated on a 1% agarose gel in TBE buffer. (C) Cleavage assay for MtuRecA. One hundred nanograms of linearized substrate 1 was incubated with either 2.5 μg of the crude extract of MtuRecA (+) or 2.5 μg of a crude extract of nontransformed E. coli BL21(De3)(pLysS) (−), for 1 h at 37°C, in 10 mM Tris-HCl (pH 8) buffer containing 10 mM MgCl₂ and 25 mM KCl. The reaction mixture was analyzed on a 1% agarose gel in TBE buffer.