V(D)J recombination assembles Igh and Igk variable region exons from hundreds of gene segments across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination in pre B cells after capturing JH-RSSs in the JH-RSS-based recombination center (RC) and then scans upstream D- and VH chromatin linearly presented by cohesin-mediated loop extrusion. During scanning, RAG utilizes D- or VH-RSSs in convergent (deletional) orientation with JH-RSSs. Igk contains four Jks (Jk1,2,4,5) and over 100 Vks in clusters oriented for deletional or inversional joining. Primary Vk-to-Jk1 joining occurs first. With loop extrusion moving deletional and inversional oriented Vks locus-wide past the Cer/Sis CBE-based diffusion platform for short-range diffusional presentation to the closely linked Jk1-based primary RC. To achieve diffusion-mediated Vk-to-Jk1 joining, Igk evolved powerful Vk and Jk RSSs. Secondary Vks rearrangements to Jks2,4,5 replace primary VkJk1 rearrangements, expanding Igk repertoires and mediating central tolerance via receptor editing.

After reviewing the Igh and primary Igk V(D)J recombination mechanism, we will present studies that elucidate the secondary Igk recombination mechanism. Primary deletional and inversional VkJk1 joins delete or displace Cer/Sis, creating a pre-B cell population that harbors secondary VkJk1-based RCs across the Vk locus and leaves most unrearranged Vks immediately upstream of secondary RCs in deletional orientation. Thus, RAG scanning from the 100-plus secondary VkJk1-based RCs, collectively, extends linearly across the Vk locus in pre-B cell populations. Studies of iPSC-generated mouse models or cell lines with physiological VkJk-rearrangements revealed that deletional and (originally) inversional Vks are mostly captured by Jk2-5-based secondary RCs in deletional orientation via linear RAG-scanning. Strong Vks-RSSs contribute to restricting secondary (editing) rearrangements to Vks immediately upstream of the RC and support low level linear scanning-based inversional Vk-to-Jk joining. We implicate Cer/Sis deletion/displacement as a developmental switch that converts the two-loop-based diffusional primary Igk rearrangement mechanism into a one-loop-based linear scanning secondary mechanism.