Figure 2.

The chromatin structure of a recombination substrate directly affects the accessibility of the recombinase for its DNA substrate. (A) Schematic diagram of the LM-PCR assay. DNA samples were ligated to blunt linkers and were subjected to PCR by using a linker primer and a second primer internal to the Rag-dependent double-strand break (arrows) at the 12 RSS (black triangle). The amplified products were subjected to Southern blot hybridization and were probed with an oligonucleotide internal to the amplified product (rectangle). (B) Measurement of double-strand break formation. Cell lines stably maintaining pSC1 or pSC1 ME were cotransfected with Rag expression vectors and were treated with drugs as indicated (lanes 1–8). Titration analysis was performed in parallel to verify the linearity of the assay (lanes 9–12). All samples were generated from the same experiment and gel. Assays were performed as described in A.