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Replication protein A interacts with AID to promote deamination of somatic hypermutation targets

Abstract

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA deaminase required for somatic hypermutation (SHM) and class switch recombination of immunoglobulin genes. Class switch recombination involves transcription through switch regions, which generates ssDNA within R loops. However, although transcription through immunoglobulin variable region exons is required for SHM, it does not generate stable ssDNA, which leaves the mechanism of AID targeting unresolved. Here we characterize the mechanism of AID targeting to in-vitro-transcribed substrates harbouring SHM motifs. We show that the targeting activity of AID is due to replication protein A (RPA), a ssDNA-binding protein involved in replication, recombination and repair. The 32-kDa subunit of RPA interacts specifically with AID from activated B cells in a manner that seems to be dependent on post-translational AID modification. Thus, our study implicates RPA as a novel factor involved in immunoglobulin diversification. We propose that B-cell-specific AID–RPA complexes preferentially bind to ssDNA of small transcription bubbles at SHM ‘hotspots’, leading to AID-mediated deamination and RPA-mediated recruitment of DNA repair proteins.

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Figure 1: Transcription-coupled deamination on SHM substrates.
Figure 2: Strand-specific targeting of AID and CF.
Figure 3: AID interacts with CF.
Figure 4: The complementing factor is RPA.
Figure 5: Coordinate binding of AID and RPA to transcribed SHM substrate.
Figure 6: B-cell specificity of the AID–RPA complex.

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Acknowledgements

We thank T. Honjo for providing AID-deficient mice and B. Stillman for providing bacterial strains expressing recombinant AID. We also thank M. Tian, A. Zarrin, J. Manis and C. Bassing for critical reading of the manuscript, D. Chowdhury for advice on the ChIP experiments, and B. Stillman and S. Elledge for discussions. This work was supported by an NIH grant to F.W.A. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

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Correspondence to Frederick W. Alt.

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Supplementary information

Supplementary Figure 1

Purification of AID. (PPT 2597 kb)

Supplementary Figure 2

The RGYW sequence undergoes preferential deamination. (PPT 62 kb)

Supplementary Figure 3

AID·RPA interaction is not mediated via DNA. (PPT 74 kb)

Supplementary Figure 4

Phosphatase treatment of AID inhibits RPA-mediated dsDNA deaminase activity. (PPT 24 kb)

Supplementary Figure 5

Dephosphorylation of AID impairs interaction with RPA. (PPT 18 kb)

Supplementary Figure 6

Chromatin association of RPA and AID at transcribed S regions. (PPT 1221 kb)

Supplementary Figure 7

Model for RPA/AID activity in SHM and CSR. (PPT 32 kb)

Supplementary Figure legends (DOC 24 kb)

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Chaudhuri, J., Khuong, C. & Alt, F. Replication protein A interacts with AID to promote deamination of somatic hypermutation targets. Nature 430, 992–998 (2004). https://doi.org/10.1038/nature02821

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