The Journal of Experimental Medicine
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Published online
doi:10.1084/jem.20062032
The Journal of Experimental Medicine, Vol. 204, No. 1, 181-190
The Rockefeller University Press, 0022-1007 $30.00
© Ronai et al.
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ARTICLE

 Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation

Diana Ronai1, Maria D. Iglesias-Ussel1, Manxia Fan1, Ziqiang Li1, Alberto Martin1,2, and Matthew D. Scharff1

1 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461
2 Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

CORRESPONDENCE Matthew D. Scharff: scharff{at}aecom.yu.edu

After encounter with antigen, the antibody repertoire is shaped by somatic hypermutation (SHM), which leads to an increase in the affinity of antibodies for the antigen, and class-switch recombination (CSR), which results in a change in the effector function of antibodies. Both SHM and CSR are initiated by activation-induced cytidine deaminase (AID), which deaminates deoxycytidine to deoxyuridine in single-stranded DNA (ssDNA). The precise mechanism responsible for the formation of ssDNA in V regions undergoing SHM has yet to be experimentally established. In this study, we searched for ssDNA in mutating V regions in which DNA–protein complexes were preserved in the context of chromatin in human B cell lines and in primary mouse B cells. We found that V regions that undergo SHM were enriched in short patches of ssDNA, rather than R loops, on both the coding and noncoding strands. Detection of these patches depended on the presence of DNA-associated proteins and required active transcription. Consistent with this, we found that both DNA strands in the V region were transcribed. We conclude that regions of DNA that are targets of SHM assemble protein–DNA complexes in which ssDNA is exposed, making it accessible to AID.

Abbreviations used: AID, activation-induced cytidine deaminase; CSR, class-switch recombination; dC, deoxycytidine; GC, germinal center; NP, 4-hydroxy-3-nitrophenyl acetyl; RNAP II, RNA polymerase II; SHM, somatic hypermutation; ssDNA, single-stranded DNA.

D. Ronai's present address is Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309.


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