Figure 1. Purification of the Signal-End Complex (SEC).

(A) Schematic diagram of the SEC purification procedure: i. Biotinylated 12RSS was bound to streptavidin agarose. ii. The paired complex was then formed in-column by the addition of core RAG proteins (MR1/MR2), HMGB1, and 23RSS DNA. iii. Unbound protein and DNA were washed out. iv. Cleavage of the DNA was initiated by Mg²⁺ and the SEC was eluted.

(B) The purification procedure was monitored by Western blot using anti-MBP. The majority of the RAG proteins did not form a complex as seen in the flow through (FT). The column was washed until no protein was detected in the FT. Following cleavage, MR1 and MR2 were present in the eluted fraction, which could be concentrated 10-fold (Conc) by ultrafiltration.

(C) The SEC was visualized on a native 7% Tris-acetate polyacrylamide gel by Coomassie staining. The corresponding band was found to contain DNA when the SEC was prepared with radio-labeled RSSs. Free DNA 12*/23* migrated close to the dye front.

(D) A 15% TBE-Urea denaturing gel showed that the concentrated eluate (Conc) contained contaminating uncut RSS but the band excised from the native gel contained a 1:1 ratio of 12 and 23 signal-ends.

(E) The SEC retains transposition activity. Strand-transfer of radio-labeled signal ends into plasmid DNA was detected on an agarose gel as one-end (nicked plasmid, faint upper band) and two-end (linearized plasmid, predominant lower band) transposition products (tnp). The SEC eluate was more efficient at transposition than the control cleavage reaction prepared using the same DNA concentrations (1 nM) and same protein concentrations (2 nM) or 10-fold more protein (20 nM).