Figure 1

Identification of bps6197 as a novel breakpoint in pGG49. (A) Plasmid substrates cleaved by the RAG proteins in vitro are subjected to linker ligation followed by PCR using a linker primer (LP) and a primer proximal to the 23-RSS (23P) to detect signal end breaks (SEBs) at the 23-RSS (large filled triangle) or another adventitious site (open stippled triangle) upstream of the 12-RSS (small open triangle). (B) pGG49 (12/23) or its derivatives containing Ttg-1 or Hox11 sequences in place of the 23-RSS (12+Ttg-1 and 12+Hox11, respectively) were incubated with WT or D600A cMR1/cMR2 and HMGB1 in an in vitro cleavage reaction and SEBs were detected by ligation-mediated PCR (LM-PCR) as described in (A). The positions of LM-PCR products revealed on an agarose gel corresponding to SEBs at the 23-RSS or a novel sequence (bps6197) are indicated at right. (C) The bps6197sequence is shown aligned to the 23-RSS in pGG49. Nucleotides in bps6197 identical to the consensus 23-RSS heptamer and nonamer sequences are underlined. The site of cleavage is identified by an arrow. A dyad-symmetric inverted repeat at the heptamer-coding junction of bps6197 is also indicated.