AID mediates hypermutation by deaminating single stranded DNA - PubMed (original) (raw)

AID mediates hypermutation by deaminating single stranded DNA

Sarah K Dickerson et al. J Exp Med. 2003.

Abstract

Activation-induced deaminase (AID) is a protein indispensable for the diversification of immunoglobulin (Ig) genes by somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion. To date, the precise role of AID in these processes has not been determined. Here we demonstrate that purified, tetrameric AID can deaminate cytidine residues in DNA, but not in RNA. Furthermore, we show that AID will bind and deaminate only single-stranded DNA, which implies a direct, functional link between hypermutation and transcription. Finally, AID does not target mutational hotspots, thus mutational targeting to specific residues must be attributed to different factors.

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Figures

Figure 1.

Figure 1.

Minimally tagged, strepAID is active in vivo and assembles into a tetramer in vitro. (a) DT40aid−/− cells can recover gene conversion activity after infection with strepAID virus, but not after infection with GST-AID virus (assay as per reference 8). (b) Phosphocellulose-purified recombinant strepAID runs with the predicted size of a tetramer. Left: Western with anti-AID antibody (reference 10). Right: silver stained gel. (c) Western blot which shows that AID tetramer (gray arrow) can be dissociated into trimer and monomer (black arrows) upon boiling in buffer containing denaturants and EDTA (lanes 2–4) but not in standard sample buffer (lane 1) or in EDTA alone (20 mM EDTA and 3% β-mercaptoethanol, lane 5). The molecular weight assignments for trimers and monomers is based on their relative position on the denaturing gel compared with protein standards. Lanes 2–4 show samples boiled in: 50% formamide and 50 mM EDTA (lane 2), 0.8 M urea and 50 mM EDTA (lane 3), 25 mM Tris (pH 8), 10 mM EDTA, and 2% SLS (lane 4).

Figure 2.

Figure 2.

Recombinant strepAID preferentially binds ssDNA. (a) EMSA: strepAID can bind single stranded DNA and RNA but does not bind double stranded DNA. (b) EMSA: an active site mutant of AID (E58Q/C87A/C90A) retains some ability to bind RNA (compare lanes 1 and 2) but cannot bind single stranded DNA (lanes 5, 7, and 9). Binding of strepAID is resistant to a 25-fold excess of tRNA (lane 4) or polydIdC (lane 6). (c) AID can be uv-cross-linked to RNA and ssDNA, and the cross-linked species run with the predicted size of an AID tetramer (closed triangle). Mutant AID can be uv-cross-linked to RNA, and the bacterial cytidine deaminase cdd can be cross-linked weakly to DNA (open triangle). (d) GST-AID can be uv-cross-linked to RNA but not to ssDNA or dsDNA (closed triangle).

Figure 3.

Figure 3.

Recombinant strepAID can deaminate ssDNA. (a) Schematic of the SNuPE reaction used to detect dC-to-dU conversion on oligonucleotide substrates. (b) The SNuPE reaction is specific: given a template with a uridine at a known position, Bst polymerase will extend a primer with the addition of the complementary base (α-P32dATP), but not with any other deoxynucleotide (α-P32dTTP, α-P32dGTP, or α-P32dCTP). (c) StrepAID-treatment of a template containing a single dC can lead to its deamination to dU. This conversion event requires active strepAID (duplicate lanes 5 and 6), and does not happen when the template is incubated with BSA (duplicate lanes 1 and 2) or with a strepAID active site mutant (E58Q/C87A/C90A, duplicate lanes 3 and 4). In addition, dsDNA is not deaminated by strepAID (lanes 7 and 8). Right panel: SNuPE of U-containing oligos serves as a calibration curve (lane 12 = signal from 100% U-containing template, lane 11 = signal from 10% U template and 90% C template, lane 10 = signal from 1% U template-99% C template, lane 9 = C-template alone).

Figure 4.

Figure 4.

Recombinant strepAID does not display hotspot preference. StrepAID deaminates a single C in the context of a T

C

G hotspot or an A

C

C coldspot. In these reactions, the C-to-U deamination efficiency is equivalent (∼1% to 5% of templates are deaminated). The experiments shown are done in duplicate using independent protein preparations. Left panel: SNuPE of U-containing oligos as a calibration curve (lane 1 = signal from 10% U-containing template and 90% C-containing template, lane 2 = signal from 1% U template-99% C template, lane 3 = C-template alone).

References

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