Fig 8.

Chi stimulates UV-dependent crosslinking of RecC to RecB. (a) RecBC^F287BpaD enzyme was reacted with Chi⁰ or Chi⁺ DNA. Reactions were started by addition of ATP and were irradiated at 259 nm from 15 s to 75 s. Samples were prepared and analyzed as in Fig 2. (b) Reactions as in (a), using the buffers (20 mM) shown. [Mg²⁺] was 5 mM except for samples marked with an asterisk, which contained 8 mM Mg²⁺. Reactions, in sets of three, were without DNA, with Chi⁰ DNA, and with Chi⁺ DNA. Additional data are in Figs. S3 and S4. (c) A surface view of a portion of RecBCD enzyme (PDB file 3K70); the left panel is an expansion of part of the right panel, which shows the entire RecBCD enzyme (Fig. 1). The RecB helicase domain is orange, and its nuclease domain is gray. RecC is blue, and RecD is green. RecC amino acid F287 (magenta) shows the position of the Bpa in RecBC^F287BpaD and the adjacent chymotrypsin cleavage site (Fig. 5) [19]. RecC amino acid F277 (red) shows the position of Bpa RecBC^F277BpaD and the nearby trypsin cleavage site (between amino acids 278 and 279). RecB amino acids A108 and R119, the most likely locations of the Bpa crosslink in RecBC^F277BpaD, are cyan and green, respectively. The measured distances between the C alpha of F277 and A108 or R119 are indicated by red arrows.