Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled

Abstract

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.

Fig. 1.

pKM2 is an effective substrate for AID. (A) The pKM2 map. The start codon in the Amp^r gene was mutated to ACG. Hypermutable regions 1 and 2 are indicated, together with the numbers of mutations and mutated clones in these regions. Black arrows indicate other mutation sites, and the numbers give the mutation positions. The numbers shown in bold are in the WRC/GYW motif. Clone numbers are given in parentheses. (B) Bacteria carrying pKM2 do not grow on carbenicillin plates. pKM2 is identical with pKM1, except that in pKM2, the Amp^r gene start codon has been changed from ATG to ACG.

Fig. 2.

AID targets hypermutable regions 1 and 2. The top lines in A and B are unmutated sequences. (A) Mutations in hypermutable region 1. Clones 1–11 grew under carbenicillin selection and clones 12–37 grew under kanamycin selection. The underlined triplets are the WRC/GYW motif, and the start codon is shown in bold. (B) Mutations in hypermutable region 2. Clones 1, 5, 6, and 8 are from carbenicillin selection, and clones 20 and 22 are from kanamycin.

Fig. 3.

Treatment of pKM2 with Topo I. (A) M, marker. Lane 1, plasmid treated with Topo I (before purification of DNA); lane 2, the same treated plasmid (after purification of DNA); and lane 3, supercoiled plasmid (not treated with Topo I). (B) SnaB1. M, marker. Lane 1, supercoiled plasmid, 100%; lane 2, plasmid treated with SnaB1, 100%; and lanes 3–7, supercoiled plasmid, 20%, 10%, 3%, 1%, and 0.5% of amount shown in lanes 1 and 2.

Fig. 4.

Alignment of SHM hotspots (Top), cytosines (Middle), and mutations (C→ T and G→ A) (Bottom) in the pKM2 plasmid. The horizontal boxes in Top and Middle are plasmid regions, as shown in Fig. 1 A. The bottom shows mutations recorded in Figs. 1 A and 2 of the entire plasmid sequence of supercoiled pKM2 treated with AID. The lowest mark represents one point mutation, and the highest mark represents 10 mutations at a given position.