Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2.  Diversity gene segments (D_H) rearrange first, followed by variable (V_H) gene rearrangements.  Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments.  D_H gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination.  In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, V_H and D_H gene segments can recombine with each other by both deletion and inversion of intervening DNA.  We propose that V_H gene segments find their targets by diffusion-controlled mechanisms.  These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.

DJ_H/VD_H recombination assays were carried out as previously described.  Genomic DNA was purified from sorted bone marrow pro-B cells (IgM^-B220⁺CD43⁺) from WT or IGCR1^-/- mice.  5-fold serial dilutions of genomic DNA (200ng, 40ng, 8ng) were used to perform PCR to analyze DJ_H rearrangements.  Primers used in this assay are listed in Table S5.  Primers flanking the ROSA26 gene were used as a loading control under the same conditions.  GeneRuler 100 bp Plus (Thermo Fisher Scientific, #SM0324) was used to confirm sizes of PCR products.

RAG2-deficient pro-B cell lines, CBE^-/-(1), CBE^-/-(2), WT, CBE^-/-(1)/C1^-/-#1, CBE^-/-(1)/C1^-/- #2, and CBE^-/-(1)/C1^-/-C2^-/-, were infected with RAG2-expressing lentivirus, and cultured in complete medium with puromycin (Thermo Fisher Scientific, #A1113803, 2μg/ml).  After 14 days selection with puromycin, cells were harvested.  Genomic DNA was collected with DNeasy Blood & Tissue Kit (Qiagen, #69506), and the DNA was used to analyze DJ_H/VDJ_H rearrangements with HotStarTaq DNA Polymerase (Qiagen, #203205) as described as above. Amplification products were analyzed by agarose gel electrophoresis. 

Raw images are corresponded to Figures 2, 3, 5, S2 and S5 in the manuscript.