Schematic representation of the DNA substrates used in the cleavage assay (not drawn to scale). ³²P incorporation on the 5′ and 3′ ends of each probe is indicated as asterisks. 12 and 23 recombination signal sequences are shown as 12 RSS and 23 RSS, respectively. Note that the 262-bp product contains one ³²P molecule and the 571 bp has two ³²P molecules, resulting in a twofold higher signal from the 571-bp fragment.

Effects of DNA-bending proteins on RAG-mediated cleavage activity. (A) Activity of purified RAG1/RAG2 (R1/2) on the 12/23 substrate, in the absence or presence of purified high mobility group protein 1 or 2 (HMG1 or HMG2, respectively), recombinant HMG1 (rHMG1), integration host factor (IHF), and HU protein. Size markers are as in previous figures. (B) Activity of purified RAG1/RAG2 (R1/2) on the 12/12 substrate, in the absence or presence of purified HMG1 or HMG2, rHMG1, IHF, and HU protein. (C) Activity of purified RAG1/RAG2 (R1/2) on 23/23 and isolated 23 RSS (23i) substrate, in the absence or presence of rHMG. Size annotations to the right of the figure refer to the 23i probe.

Observation of 12/23-regulated cleavage by whole cell extract. (A) Cleavage activity of RAG1/ RAG2-cotransfected 293T whole cell extracts (R1/2 WCE) on 12/23 and 12/12 cleavage probes. (B) Cleavage activity of RAG1/RAG2-cotransfected 293T whole cell extract (R1/2 WCE) on 12/23 and 23/23 cleavage probes. (C) Cleavage activity of copurified RAG1/RAG2 (R1/2) on 12/23 and 12/12 cleavage probes. The 900-bp product resulting from uncoupled cleavage at the 12 RSS is annotated. Asterisks indicate cryptic cleavage sites. (D) Complementation experiment with purified RAG1/RAG2 (R1/2) and untransfected 293 whole cell extract (WCE), using 12/23 and 12/12 substrate. (E) Cleavage activity of purified RAG1/RAG2 (R1/2) on 12/23 and 23/23 cleavage probes, in the presence or absence of untransfected 293T whole cell extract (WCE), as indicated. All samples were resolved on 4.5% native polyacrylamide gels and subsequently autoradiographed. Positions of the uncleaved 1151-bp 12/12 substrate, the uncleaved 1,172-bp 23/23 substrate, and the uncleaved 1,162-bp 12/23 substrate are indicated. The 571 and 262 bp annotations indicate the positions of the expected coupled cleavage products in all cases. The size and migration of all the cleavage products were independently confirmed by restriction analysis of the substrate. Radiolabeled 1-kb size marker was used in each experiment as an independent size marker. All volumes of protein and extracts used are indicated in microliters.

Combined effects on RAG-mediated cleavage by untransfected 293 WCE, rHMG1, and purified RAG1/RAG2. (A) Activity of indicated combinations of R1/2, rHMG1, and WCE on the 12/23 substrate. Size markers are as indicated in previous figures. (B) Activity of R1/2, rHMG1, and WCE on the 12/12 substrate. (C) Activity of R1/2, rHMG1, and WCE on the 23/23 substrate. (D) Activity of R1/2, rHMG1, and WCE on the isolated 23RSS substrate.