Despite four decades of study, major gaps remain in our understanding of key molecular and cellular processes that orchestrate the action of the RAG1/RAG2 endonuclease during antigen receptor (AgR) gene assembly by V(D)J recombination. V(D)J recombination occurs in focal regions of RAG binding in AgR loci known as recombination centers (RCs) but virtually nothing is known about the composition or organizing principles of RCs. Our recent findings demonstrate that recombinase function is modulated by nuclear compartmentalization of RAG1 and that RAG1's large N-terminal region (NTR) controls RAG activity, trafficking, chromatin binding, gene segment repertoire, and interaction with a newly discovered recombination factor, Spin1. However, RAG nuclear cell biology is poorly understood and the RAG1 NTR is largely a black box, with almost no understanding of its structure, the residues that mediate its many functions, or how it works together with the remainder of the RAG enzyme. Indeed, no structures are available for the holo-RAG complex, severely limiting insight into cooperation between the catalytic and regulatory domains. The central objective of our proposed experiments is to fill these gaps in understanding through a systematic dissection of RCs and RAG nuclear cell biology, the function of the RAG1 NTR, and the structure of the holo-RAG-nucleosome complex. We will use complementary biochemical, imaging, molecular, structural, and genetic approaches to achieve the following aims: Aim 1. Delineate RAG nuclear cell biology and the contribution of the RAG1 NTR. Our findings reveal a repressive association of RAG1 with nucleoli and repositioning of RAG1 adjacent to nuclear speckles when recombination is activated. We will systematically characterize RAG nuclear compartmentalization, function, and entry into RCs, determine how each parameter is controlled by binding partners and the RAG1 NTR, and establish the physiological role of RAG1 nucleolar association in a novel RAG1 mutant mouse. Aim 2. Determine components of and regulatory mechanisms in RCs. We will identify the components of RCs using in vivo proximity labeling and quantitative proteomics, establish the mechanism by which Spin1 interacts with RAG1 and stimulates recombination, and determine how the RAG1 NTR dictates gene segment repertoire through control of the balance between short- and long-range modes of recombination. Aim 3. Determine the structural basis of holo-RAG-nucleosome interactions. Building on extensive preliminary data, we will use: i) X-ray crystallography to determine the structure of a RAG1 NTR fragment free and bound to histone tails; ii) cryo-electron microscopy to determine the structure of RAG1/2 bound to DNA and the nucleosome core particle; and iii) cryo-electron tomography to determine the structure of holo RAG bound to chromatin in situ in the nucleus-thereby providing unprecedented insight into the quaternary structure of RAG, its mechanism of recruitment to ch...