Fig. 3.

Further characterization of the Chi-dependent protease cleavage of RecC. (a) RecBCD enzyme was reacted with uncapped lambda Chi⁰ or Chi⁺ DNA, as in Fig. 2, or with lambda substrates capped at the left or right end of lambda, marked L and R, to block RecBCD entry at the capped end. Chi is active only if RecBCD enzyme enters from the right end [8]. Reactions were with 5 mM ATP and 2.5 mM Mg²⁺ (low Mg²⁺) or 8 mM Mg²⁺ (high Mg²⁺), as shown, and reacted with 10 nM trypsin for 1 min before Western blot analysis using RecC-specific polyclonal antibodies. (b) As in (a) but showing polypeptides detected with anti-RecB polyclonal antibodies. (c) Quantification of the Chi-dependent fragment in (a) and in parallel reactions that used 3.2 and 32 nM trypsin; x and o, Chi⁺ and Chi⁰ reactions; L and R, left- and right-end caps. (d) Persistence of RecC’s Chi-dependent sensitization to trypsin. RecBCD was reacted with Chi⁺ or Chi⁰ DNA for 1 min; the reactions were stopped by addition of EDTA to 23 mM, incubated for the further times indicated, and then treated for 1 min with 160 nM trypsin. RecC fragments were detected as in Fig. 2. Continuous and broken lines, ~91-kDa Chi-dependent fragment 1 and ~82-kDa Chi-independent fragment 2. (e) DNA substrate used in (a), (b), and (c). Phage lambda DNA (48.5 kb) with a Chi site (χ⁺D) 3.5 kb from the right end. Hairpin DNA (gray, data not drawn to scale) ligated in some cases to one strand at one or the other end of the lambda DNA substrate prevents RecBCD from unwinding from the capped end. Arrow, direction RecBCD must unwind DNA to be changed by χ⁺D [8].