Figure 3. Cleavage of cRSS by RAG proteins.

Standard, cryptic and nonspecific DNA substrates were 3’ labeled (*) on top or bottom strands. Expected nick (35nt) and nick-nick (16nt) products were run in the MW lane to compare the actual nick sizes. Hairpin products were of various sizes. Reactions included (A) an excess of unlabeled NS DNA and Mn²⁺ and (B) 23RSS and Mg²⁺. The arrow indicates the nick product. The results are representative of 7 nicking assays in the presence of Mn²⁺and 4 experiments using Mg²⁺. In the presence of Mg²⁺, the mean and SE of the cleavage product expressed as the percentage of total DNA in the lane appearing in the band of expected size (34 bp) was 4.6 ± 1.8 for NS DNA, 6.2 ± 2.2 for VH3-23, 9.1 ± 3.2 for VH4-59 and 62.8 ± 15.7 for 12RSS. In the presence of Mn²⁺, the mean ± SE of the cleavage product was 7.6 ± 1.9 for NS DNA, 9.4 ± 2.1 for VH3-23, 17.3 ± 4.6 for VH4-59 and 57.0 ± 7.3 for 12RSS. Significant differences were found in RAG-mediated cleavage of IGHV4-59 versus NS DNA (p=0.02) and IGHV3-23 versus IGHV4-59 (p=.03) in the presence of Mn²⁺.