Figure 7. A jawed vertebrate‐specific extended loop on RAG2 contacts target DNA and inhibits transposition.
- Comparison of RAG2 and BbeRAG2L structures. Red color indicates the amino acid 333–342 RAG2 loop and the equivalent loop of BbeRAG2. The four‐amino‐acid deletion, (ΔL in (D)), is indicated with green brackets. Due to areas of low resolution in the BbeRAG2L structure, some regions that are likely to be beta strands cannot be modeled as such.
- Sequence alignment of RAG2 and RAG2‐like proteins in the vicinity of mouse RAG2 loop 333–342. Green shading, four amino acids deleted at tip of loop. Red letters in blue box, relatively well‐conserved residues; white letters on red background, highly conserved residues.
- Cryo‐EM map (transparent) and cartoon model of RAG2 showing contact between the extended loop (red) and target site DNA (yellow; stick model).
- Results of in vivo plasmid‐to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (ΔL) the four amino acids at the tip of the 333–342 loop, performed as described in (Zhang et al, 2019); (****P < 0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.
- Results of in vivo plasmid‐to-genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated. As previously demonstrated (Zhang et al, 2019), R848 (present in WT RAG1) and RAG2 acidic hinge residues 362–383 each potently suppress transposition. Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1–361, which is further increased by ΔL; (***P = 0.001). Four biological replicates for each condition.
- In vivo recombination activity in HEK293T cells of mouse wild‐type RAG1 or RAG1 R848M with RAG2 1–361 or 1–361 ∆L. (P = 0.42 for wild‐type RAG1 group, P = 0.41 for RAG1 R848M group). Three biological replicates for each condition.