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. 2007 May 15;104(20):8409-14.
doi: 10.1073/pnas.0702836104. Epub 2007 May 8.

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

Belén Gómez-González  1 , Andrés Aguilera
Affiliations

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

Belén Gómez-González et al. Proc Natl Acad Sci U S A. .

Abstract

Activation-induced cytidine deaminase (AID) is a B cell enzyme essential for Ig somatic hypermutation and class switch recombination. AID acts on ssDNA, and switch regions of Ig genes, a target of AID, form R-loops that contain ssDNA. Nevertheless, how AID action is specifically targeted to particular DNA sequences is not clear. Because mutations altering cotranscriptional messenger ribonucleoprotein (mRNP) formation such as those in THO/TREX in yeast promote R-loops, we investigated whether the cotranscriptional assembly of mRNPs could affect AID targeting. Here we show that AID action is transcription-dependent in yeast and that strong and transcription-dependent hypermutation and hyperrecombination are induced by AID if cells are deprived of THO. In these strains AID-induced mutations occurred preferentially at WRC motifs in the nontranscribed DNA strand. We propose that a suboptimal cotranscriptional mRNP assembly at particular DNA regions could play an important role in Ig diversification and genome dynamics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effect of transcription and AID on the frequency of mutation in S. cerevisiae wild-type and THO mutant strains. (A) Frequency of Kar− mutants obtained in the GKAR1 system. (B) Frequency of Ura− mutants obtained in the LAUR system and Rad52 dependency. (C) Effect of AID on the frequency of Ura− mutants in the LAUR system in wild-type, mft1Δ, rad52Δ, and mft1Δ rad52Δ strains. All experiments were performed under high (ON) and low (OFF) transcription. Diagrams of systems are shown above each graph. Mean mutation frequency and standard deviation of three to four different fluctuation tests are plotted.
Fig. 2.
Fig. 2.
Genetic analysis of spontaneous (−AID) and AID-induced (+AID) mutations. Distribution of lacZ−:lacZ+ mutations among Ura− mutants in wild-type and mft1Δ strains is shown. An asterisk indicates a statistically significant difference (P < 0.05) with respect to the wild-type value under the same transcription conditions, as determined by contingency χ2 analysis.
Fig. 3.
Fig. 3.
Effect of AID on transcription-associated recombination. (A) Spontaneous (−AID) and AID-induced (+AID) frequency of gene conversion under high (ON) and low (OFF) transcription between a plasmid leu2-r and a chromosomal leu2-k allele in a wild-type strain. (B) Effect of AID expression on direct-repeat recombination in the GLG system. The frequency of GFP+ recombinants (signals inside the box) is indicated. y axis, green fluorescence (FL1H); x axis, unspecific fluorescence (FL2H). A diagram of each system is shown on the top of each panel.
Fig. 4.
Fig. 4.
A model to explain stimulation of genome instability by AID in yeast THO mutants compared with the S regions in B cells. (A) Transcription and mRNP formation mediated by THO in wild-type and THO-depleted yeast cells. In wild-type yeast, AID is able to act on the DNA when it is transcribed. Negative supercoiling behind the elongating RNA polymerase II (RNAPII) allows a weak AID action. In THO-depleted yeast, a cotranscriptional R-loop can be formed, allowing AID to act on the nontranscribed ssDNA, causing strong hypermutation and hyperrecombination. (B) A putative example of transcription and mRNP formation in the S region of Ig genes in human B cells. The G-rich S region could be refractory to a number of RNA-binding proteins, therefore forming a suboptimal mRNP at that particular region. Nascent RNA S regions could lead to local cotranscriptional R-loops or G-loops that would allow AID action.
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