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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\u2013AID 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.<\/jats:p>","DOI":"10.1093\/nar\/gkag806","type":"journal-article","created":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T12:02:04Z","timestamp":1785585724000},"source":"Crossref","is-referenced-by-count":0,"title":["AID self-assembly and multistranded DNA binding drive synapsis in class switch recombination"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7930-0636","authenticated-orcid":false,"given":"Di","family":"Liu","sequence":"first","affiliation":[{"name":"Department of Transfusion Medicine, The Second Affiliated Hospital of Xi\u2019an Jiaotong University , Xi\u2019an, Shaanxi 710004 ,","place":["China"]},{"name":"Institute of Molecular and Translational Medicine (IMTM), and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi\u2019an Jiaotong University Health Science Center , Xi\u2019an, Shaanxi 710061 ,","place":["China"]},{"name":"Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education , Xi\u2019an, Shaanxi 710061 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Translational Medicine (IMTM), and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi\u2019an Jiaotong University Health Science Center , Xi\u2019an, Shaanxi 710061 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuhang","family":"He","sequence":"additional","affiliation":[{"name":"Beijing National Laboratory for Condensed Matter Physics and Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences , Beijing 100190 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongtao","family":"Zhu","sequence":"additional","affiliation":[{"name":"Beijing National Laboratory for Condensed Matter Physics and Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences , Beijing 100190 ,","place":["China"]},{"name":"Department of Physics, University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keyin","family":"Qi","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Translational Medicine (IMTM), and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi\u2019an Jiaotong University Health Science Center , Xi\u2019an, Shaanxi 710061 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanying","family":"Dong","sequence":"additional","affiliation":[{"name":"Department of Transfusion Medicine, The Second Affiliated Hospital of Xi\u2019an Jiaotong University , Xi\u2019an, Shaanxi 710004 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5894-0079","authenticated-orcid":false,"given":"Bailin","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Transfusion Medicine, The Second Affiliated Hospital of Xi\u2019an Jiaotong University , Xi\u2019an, Shaanxi 710004 ,","place":["China"]},{"name":"Institute of Molecular and Translational Medicine (IMTM), and Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi\u2019an Jiaotong University Health Science Center , Xi\u2019an, Shaanxi 710061 ,","place":["China"]},{"name":"Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education , Xi\u2019an, Shaanxi 710061 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