ABSTRACT Diversification of our immune system requires two primary DNA recombination pathways: V(D)J and class switch recombination (CSR). Both V(D)J and CSR have several poorly understood intermediate steps. These intermediate steps are the basis for the most disease-relevant aspects of these pathways because they are inherently unstable. Static structural biology approaches alone are not sufficient to understand the instability of these intermediates. The dynamic approaches described here permit us to understand these unstable intermediates that are key to both inherited and acquired (neoplastic) diseases of the V(D)J and CSR pathways. From an applied standpoint, the understanding gained in this proposal positions us to eventually use biochemical systems to generate improved antibodies against pathogenic viruses and bacteria. Important for the current proposal, over 85% of human lymphoid malignancies are B cell in nature, and we have shown that the breakage phase at the two chromosomes arises by a confluence of failures in the V(D)J and Ig CSR mechanisms. The chromosome break at the immunoglobulin locus is typically due to failures during the synapsis steps as the RAG complex prematurely releases the ends. Failures can also occur in the RAG hand- off to the NHEJ pathway (for joining the ends). We study all of these aspects of RAG function in this proposal. The other chromosome break arises due to the off-target behavior of the CSR enzyme called activation-induced deaminase (AID), which we study in the second Project of this proposal. The Lieber lab has done key biochemistry on all of the enzymes mentioned above. We are the first and only lab to reconstitute the entire V(D)J pathway using fully purified enzymes. Despite beautiful recent atomic structures of RAG and AID proteins, the dynamic action of these enzymes and how they fail is the gap that remains. In addition to neoplasms, diseases caused by RAG and AID enzymes are responsible for over one-third of inherited human immune deficiencies called SCID. My lab has used the current funding period to develop in-lab capability to use our unique purified proteins for V(D)J and CSR in high resolution single molecule assays, specifically cryo-EM and sm-FRET. In 2019 and 2020, we published the first sm-FRET in which not only the proteins but also the dynamic sm-FRET were done in my lab. My lab also now has full cryo-EM abilities, which would be relevant at the later stages of the current proposal. We also can carry out the relevant biochemical steps in this proposal on mono- and polynucleosomal substrates in addition to naked DNA. In Project 1, we dissect the key vulnerable points in the RAG synapsis steps and their hand-off to the NHEJ pathway. In Project 2, we study the independent process of Ig class switch recombination (CSR). The Lieber lab was the first to discover kilobase length chromosomal R-loops at switch sequences. We are the only lab able to reconstitute the entire CSR pathway using puri...