AID self-assembly and multistranded DNA binding drive synapsis in class switch recombination

Activation-induced deaminase (AID) initiates immunoglobulin class switch recombination (CSR) by deaminating cytosines within transcription-generated single-stranded DNA in switch regions. R-loops formed during switch-region transcription are thought to expose AID substrates; however, how AID engages and organizes these complex nucleic acid structures remain unclear. Here, combining ensemble biochemistry with single-molecule colocalization and fluorescence resonance energy transfer (FRET) analyses, we uncover an unexpected role for AID as a DNA synapsis factor. AID preferentially promotes synapsis between multistranded DNA substrates, including R-loops and tailed D-loops, and stabilizes these higher-order synaptic complexes. Mutational analyses reveal that two distinct nucleic acid-binding pockets cooperate to drive efficient synapsis. Single-molecule FRET further reveals that AID promotes intramolecular synapsis of tailed D-loops that mimic key CSR intermediates. Moreover, three-color single-molecule analyses indicate that DNA binding-associated AID self-assembly, consistent with AID assemblies observed in cells, accompanies with synaptic complex formation. Notably, a catalytically active AID mutant with impaired AID-AID interactions shows severely compromised DNA synapsis, indicating that higher-order AID organization is essential for synaptic complex formation and synapsis is mechanistically separable from cytosine deamination. Together, our findings establish AID as a DNA synapsis factor and support a model in which AID self-assembly and multistranded DNA binding drive higher-order synapsis during CSR.