FIG. 1.
Synthetic hairpin nicking mediated by the RAG proteins. (A) Synthetic hairpin nicking occurs in the presence of Mn2+ but not Mg2+. Annealed 5′- and 3′-end-labeled hairpin oligonucleotides were incubated with truncated, coexpressed MH- or GST-tagged RAG proteins. Sites of nicking distant from the hairpin tip (small arrows and lowercase letters) and 58-nt (5′ labeled) and 68-nt (3′ labeled) fragments corresponding to nicking at a position 5 nt 5′ of the hairpin tip (large arrows) are shown. Reactions included either 10 mM Mg2+ (Mg), 1 mM Mn2+ (Mn), or 10 mM EDTA (E). Positions of DNA molecular weight markers (M) are indicated in nucleotides. An asterisk is used to indicate the site of 32P end labeling. (B) High-resolution mapping of synthetic hairpin nicking products. Samples from lanes 3, 6, 10, and 13 of panel A were reanalyzed adjacent to marker fragments by denaturing PAGE. The 58- and 68-nt reciprocal products of hairpin nicking generated with 5′- and 3′-end-labeled substrates, respectively, are indicated with arrows.