52 / 2026-03-31 20:02:40

Linear RAG Scanning Mediates Editing of Igκ Variable Region Repertoires

V(D)J recombination,receptor editing,Linear RAG Scanning,Loop extrusion,antibody

V(D)J recombination-mediated Igκ light chain variable region exon assembly in precursor (pre)-B cells involves RAG endonuclease-orchestrated cleavage between and joining of paired Vκ and Jκ gene segments and flanking RAG-targeting recombination signal sequences (“RSSs”).  The 3.1Mb Igκ contains four Jks (Jk1,2,4,5) and 100-plus Vks in clusters oriented for deletional or inversional joining. Vk-to-Jk joining is ordered, with primary Vk-to-Jk1 rearrangements occuring first, followed by secondary rearrangements of upstream Vks that replace primary VkJk1s by joining to Jk2-5.  Loop extrusion moves deletional-oriented and inversional-oriented, locus-wide Vks past the Cer/Sis CBE-based diffusion platform for short-range diffusional presentation to Jk1-bound RAG in the primary recombination center (RC).  To achieve diffusion-mediated Vk-to-Jk1 joining, Igk evolved powerful Vk- and Jk-associated RSSs.  Secondary Igkrearrangements replace non-functional or autoreactive primary VkJk1 rearrangements, expanding the Igk repertoire and mediating central tolerance via receptor editing.  Here, we describe studies that elucidate the physiologically-critical secondary Igk recombination mechanism. Primary deletional and inversional VκJκ1 joins, respectively, delete or displace Cer/Sis, creating a pre-B cell population that harbors secondary VκJκ1-based RCs across the Vκ locus and leaves most unrearranged Vκs immediately upstream of secondary RCs in deletional orientation. High throughput assays demonstrated that RAG scanning from secondary VkJk1-based RCs, collectively, extends linearly across the Vk locus in primary pre-B cell populations. Correspondingly, studies of iPSC-generated mouse models or cell lines with physiological VkJk-rearrangements further revealed that deletional and, originally, inversional Vκs are mostly captured by Jκ2-5-based secondary RCs in deletional orientation via linear RAG-scanning. Strong Vκ-RSSs contribute to restricting secondary rearrangements, including potential editing rearrangements, to Vks immediately upstream of a given secondary RC and support, at lower level, linear scanning-based inversional Vk-to-Jκ rearrangements. Our findings 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 rearrangement mechanism.