Skip to main content
. 2006 Oct 24;4(11):e350. doi: 10.1371/journal.pbio.0040350

Figure 5. The CT Hairpin Inhibits Target Capture.

Figure 5

(A) CT hairpin is bound weakly by the RAG–signal end complex and inhibits target capture of a GC target. Using labeled CT or GC target, target complex formation was detected on a native 4%–20% gradient acrylamide gel (lanes 3 and 4). Unlabeled CT hairpin was mixed in a 1:1 molar ratio with labeled GC target prior to start of reaction (lane 5).

(B) GC target is bound by the RAG-signal end complex. CT hairpin is bound only weakly by the RAG–signal end complex and inhibits target capture of a GC target. Using labeled GC or CT target, complex formation was detected on a native 4%–20% gradient acrylamide gel. Proteinase K treatment indicates amount of RAG-bound transposition products present in DNA-protein complex. Unlabeled CT hairpin was mixed with labeled GC target prior to start of reaction (lanes 5 and 6).

(C) CT hairpin destabilizes preformed GC target capture complexes. Radiolabeled GC target was bound to RAG–signal end complexes for 120 min, upon which non-labeled CT inhibitor was added to reaction. Reaction was stopped after an additional 30, 60, or 120 min, as indicated.

(D) CT hairpin does not destabilize signal end complexes formed by cleavage. RAG proteins were incubated for 3 h with PCR-generated cleavage substrate. After the reaction, either stop buffer (containing proteinase K and SDS) or CT inhibitor was added. Expected cleavage products are indicated to right of gel; arrowheads indicate single cleavage products.