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AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants.

Ruiz JF, Gómez-González B, Aguilera A - PLoS Genet. (2011)

Bottom Line: We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ).AID-induced translocations were strongly enhanced in yeast THO mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation.Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, Sevilla, Spain.

ABSTRACT
Transcription of the switch (S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID), triggering class switch recombination (CSR), as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ). AID-induced translocations were strongly enhanced in yeast THO mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

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Intron-based assay for chromosomal translocations in yeast.(A) Scheme of the translocation system used. There is no sequence homology between the two DNA constructs integrated in chromosomes III and XV. Chromosomal translocations detected generate a galactose-inducible full-length LEU2 gene harboring a yeast ACT1 intron sequence inside. Size of chromosomal fragments is indicated. (B) Leu+ translocation frequencies after HO-induced DSBs in both wild-type and hpr1Δ cells. The presence (w) or absence (w/o) of the Sµ350 sequence and AID overexpression are indicated. Translocation frequencies were obtained as the median value of at least ten independent colonies for each strain. Median values and the corresponding standard deviations are shown. (C) Molecular karyotype of Leu+ translocants analyzed by PFGE. Agarose gels were stained with ethidium bromide (left) and analyzed by Southern using a radiolabeled LEU2 probe (right). The electrophoretic mobility of natural yeast chromosomes is indicated. The LEU2 signal in parental cells corresponds to the two non-homologous halves of LEU2 integrated in chromosomes III and XV (bold). Chromosomes XV and VII have the same electrophoretic mobility in the experimental conditions used here. The translocated chromosomes containing LEU2 (tIII/XV) or not containing it (tXV/III), are marked with black triangles, and their sizes are indicated.
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pgen-1002009-g002: Intron-based assay for chromosomal translocations in yeast.(A) Scheme of the translocation system used. There is no sequence homology between the two DNA constructs integrated in chromosomes III and XV. Chromosomal translocations detected generate a galactose-inducible full-length LEU2 gene harboring a yeast ACT1 intron sequence inside. Size of chromosomal fragments is indicated. (B) Leu+ translocation frequencies after HO-induced DSBs in both wild-type and hpr1Δ cells. The presence (w) or absence (w/o) of the Sµ350 sequence and AID overexpression are indicated. Translocation frequencies were obtained as the median value of at least ten independent colonies for each strain. Median values and the corresponding standard deviations are shown. (C) Molecular karyotype of Leu+ translocants analyzed by PFGE. Agarose gels were stained with ethidium bromide (left) and analyzed by Southern using a radiolabeled LEU2 probe (right). The electrophoretic mobility of natural yeast chromosomes is indicated. The LEU2 signal in parental cells corresponds to the two non-homologous halves of LEU2 integrated in chromosomes III and XV (bold). Chromosomes XV and VII have the same electrophoretic mobility in the experimental conditions used here. The translocated chromosomes containing LEU2 (tIII/XV) or not containing it (tXV/III), are marked with black triangles, and their sizes are indicated.

Mentions: In order to unequivocally assay the capacity of R-loops to form DSBs, we developed a yeast system for the detection of reciprocal CTs that should occur via DSBs in the absence of DNA homology. The system is based on two non-homologous halves of the LEU2 gene (leu2Δ5′ and leu2Δ3′) integrated at chromosomes XV and III, respectively (Figure 2A). Upstream to the leu2Δ5′ at chromosome XV, an HO endonuclease cut site fused to the hygromycin gene and the 246-bp 3′-end of the ACT1 intron were integrated (HO-ACT1iΔ5’-leu2Δ5’). The leu2Δ3′ sequence, under control of the GAL1 promoter, followed by the remaining 62-bp of the ACT1 intron and the Sµ350 sequence (leu2Δ3′-ACT1iΔ3′-Sµ350) was integrated at the right arm of chromosome III together with the URA3 gene (Figure 2A). As a control we used a similar system carrying no Sµ350 sequence (leu2Δ3′-ACT1iΔ3′). These genetically engineered chromosomes III and XV were made in haploid MATa-inc cells containing the HO endonuclease gene under the GAL1 promoter, and the endogenous LEU2 gene and the ACT1 intron deleted from their chromosomal loci. In galactose, the HO endonuclease is activated causing a DSB at the HO-ACT1iΔ5’-leu2Δ5’ construct on chromosome XV, whereas the leu2Δ3′-ACT1iΔ3′-Sµ350 on chromosome III is heavily transcribed from the GAL1 promoter. Therefore, the HO-induced DSBs on chromosome XV could be sealed with a transcription-mediated DSB on chromosome III to lead a Leu+ translocations (Figure 2A). These translocations would be expected to contain the breakpoints embedded in a functional ACT1 intron located between the two LEU2 gene halves (Figure 2A).


AID induces double-strand breaks at immunoglobulin switch regions and c-MYC causing chromosomal translocations in yeast THO mutants.

Ruiz JF, Gómez-González B, Aguilera A - PLoS Genet. (2011)

Intron-based assay for chromosomal translocations in yeast.(A) Scheme of the translocation system used. There is no sequence homology between the two DNA constructs integrated in chromosomes III and XV. Chromosomal translocations detected generate a galactose-inducible full-length LEU2 gene harboring a yeast ACT1 intron sequence inside. Size of chromosomal fragments is indicated. (B) Leu+ translocation frequencies after HO-induced DSBs in both wild-type and hpr1Δ cells. The presence (w) or absence (w/o) of the Sµ350 sequence and AID overexpression are indicated. Translocation frequencies were obtained as the median value of at least ten independent colonies for each strain. Median values and the corresponding standard deviations are shown. (C) Molecular karyotype of Leu+ translocants analyzed by PFGE. Agarose gels were stained with ethidium bromide (left) and analyzed by Southern using a radiolabeled LEU2 probe (right). The electrophoretic mobility of natural yeast chromosomes is indicated. The LEU2 signal in parental cells corresponds to the two non-homologous halves of LEU2 integrated in chromosomes III and XV (bold). Chromosomes XV and VII have the same electrophoretic mobility in the experimental conditions used here. The translocated chromosomes containing LEU2 (tIII/XV) or not containing it (tXV/III), are marked with black triangles, and their sizes are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044682&req=5

pgen-1002009-g002: Intron-based assay for chromosomal translocations in yeast.(A) Scheme of the translocation system used. There is no sequence homology between the two DNA constructs integrated in chromosomes III and XV. Chromosomal translocations detected generate a galactose-inducible full-length LEU2 gene harboring a yeast ACT1 intron sequence inside. Size of chromosomal fragments is indicated. (B) Leu+ translocation frequencies after HO-induced DSBs in both wild-type and hpr1Δ cells. The presence (w) or absence (w/o) of the Sµ350 sequence and AID overexpression are indicated. Translocation frequencies were obtained as the median value of at least ten independent colonies for each strain. Median values and the corresponding standard deviations are shown. (C) Molecular karyotype of Leu+ translocants analyzed by PFGE. Agarose gels were stained with ethidium bromide (left) and analyzed by Southern using a radiolabeled LEU2 probe (right). The electrophoretic mobility of natural yeast chromosomes is indicated. The LEU2 signal in parental cells corresponds to the two non-homologous halves of LEU2 integrated in chromosomes III and XV (bold). Chromosomes XV and VII have the same electrophoretic mobility in the experimental conditions used here. The translocated chromosomes containing LEU2 (tIII/XV) or not containing it (tXV/III), are marked with black triangles, and their sizes are indicated.
Mentions: In order to unequivocally assay the capacity of R-loops to form DSBs, we developed a yeast system for the detection of reciprocal CTs that should occur via DSBs in the absence of DNA homology. The system is based on two non-homologous halves of the LEU2 gene (leu2Δ5′ and leu2Δ3′) integrated at chromosomes XV and III, respectively (Figure 2A). Upstream to the leu2Δ5′ at chromosome XV, an HO endonuclease cut site fused to the hygromycin gene and the 246-bp 3′-end of the ACT1 intron were integrated (HO-ACT1iΔ5’-leu2Δ5’). The leu2Δ3′ sequence, under control of the GAL1 promoter, followed by the remaining 62-bp of the ACT1 intron and the Sµ350 sequence (leu2Δ3′-ACT1iΔ3′-Sµ350) was integrated at the right arm of chromosome III together with the URA3 gene (Figure 2A). As a control we used a similar system carrying no Sµ350 sequence (leu2Δ3′-ACT1iΔ3′). These genetically engineered chromosomes III and XV were made in haploid MATa-inc cells containing the HO endonuclease gene under the GAL1 promoter, and the endogenous LEU2 gene and the ACT1 intron deleted from their chromosomal loci. In galactose, the HO endonuclease is activated causing a DSB at the HO-ACT1iΔ5’-leu2Δ5’ construct on chromosome XV, whereas the leu2Δ3′-ACT1iΔ3′-Sµ350 on chromosome III is heavily transcribed from the GAL1 promoter. Therefore, the HO-induced DSBs on chromosome XV could be sealed with a transcription-mediated DSB on chromosome III to lead a Leu+ translocations (Figure 2A). These translocations would be expected to contain the breakpoints embedded in a functional ACT1 intron located between the two LEU2 gene halves (Figure 2A).

Bottom Line: We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ).AID-induced translocations were strongly enhanced in yeast THO mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation.Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, Sevilla, Spain.

ABSTRACT
Transcription of the switch (S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase (AID), triggering class switch recombination (CSR), as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks (DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining (NHEJ). AID-induced translocations were strongly enhanced in yeast THO mutants, consistent with the idea that AID-mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID-mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.

Show MeSH
Related in: MedlinePlus