FIGURE 5.

RecA⋅ATP-mediated strand exchange with short internal heterology and at the 5′-end in the presence of MutSL. (A) Scheme of the reaction between css_hom and the lds_het–ins substrate with internal region toward the 3′ end and at the 5′-end (black and gray squares, respectively). The css_hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, after which RecA, (MutS, MutL or MutSL) and the lds_het–ins substrate were added and incubated (60 min, 37°C). The percentage of jm intermediates and nc products are shown beneath the gel. Lane 1, the css and lds substrates (termed C). Results shown as the mean ±5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css_hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP, followed by the 3353-bp lds_het–ins substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration, and incubated (60 min, 37°C). The reaction was separated by gel electrophoresis and quantified. The left-hand side bar denotes the fraction of jm intermediates (gray bar) and the right-hand side scale denotes the fraction of unreactive lds_het–ins substrate (black bars). The minus (–) symbol indicates lack of MutS or MutL.