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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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Analysis of recombinogenic potential of the c-MYC sequence.(A) AID-independent recombinogenic behavior of c-MYC in THO mutants. A 3.8-kb fragment of the human c-MYC, as well as the control 4.1-kb long LYS2 sequence, were cloned into the GFP-based direct-repeat recombination system. Details as in Figure 1. (B) Leu+ translocation frequencies in c-MYC-containing wild-type and hpr1Δ cells after HO-induced DSBs. Details as in Figure 2. (C) Analysis by PCR of CT breakpoints in hpr1Δ yeast at the c-MYC sequence. Breakpoints from cells with (grey) and without (black) AID-overexpression are indicated by arrows (top). The analysis of the breakpoint junctions is shown at the bottom. The c-MYC/HO cut site junctions from eight AID- and five AID+ independent Leu+ translocant strains are shown. The length of the c-MYC sequence within the reconstituted ACT1 intron in the translocant chromosome is indicated. Underlined nucleotide residues indicate microhomologies between the MYC and HO cut site sequences. The sequence downstream of the HO cleavage site is shown on top.
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pgen-1002009-g005: Analysis of recombinogenic potential of the c-MYC sequence.(A) AID-independent recombinogenic behavior of c-MYC in THO mutants. A 3.8-kb fragment of the human c-MYC, as well as the control 4.1-kb long LYS2 sequence, were cloned into the GFP-based direct-repeat recombination system. Details as in Figure 1. (B) Leu+ translocation frequencies in c-MYC-containing wild-type and hpr1Δ cells after HO-induced DSBs. Details as in Figure 2. (C) Analysis by PCR of CT breakpoints in hpr1Δ yeast at the c-MYC sequence. Breakpoints from cells with (grey) and without (black) AID-overexpression are indicated by arrows (top). The analysis of the breakpoint junctions is shown at the bottom. The c-MYC/HO cut site junctions from eight AID- and five AID+ independent Leu+ translocant strains are shown. The length of the c-MYC sequence within the reconstituted ACT1 intron in the translocant chromosome is indicated. Underlined nucleotide residues indicate microhomologies between the MYC and HO cut site sequences. The sequence downstream of the HO cleavage site is shown on top.

Mentions: Therefore, we cloned a 3.8-kb fragment of the human c-MYC gene that included the first exon and the first intron in our GFP direct-repeat recombination assay, as a functional and structural equivalent of a long S region. As can be seen in Figure 5A, whereas recombination was not affected by the presence of the MYC sequence in wild-type cells, regardless of AID expression, a strong increase was seen in hpr1Δ cells both without and with AID expression (24-fold and 32-fold increase with respect to the control wild-type, respectively; Figure 5A). To test whether these high recombination levels, notably higher than those obtained with the Sµ350 sequence (compare Figure 1 and Figure 5A), were not just due to the presence of a longer DNA sequence between the GFP repeats, we tested the effect of the long (4.1 kb) and low GC-rich (40%) sequence of the LYS2 gene. Recombination levels in this system containing the LYS2 sequence dropped markedly (Figure 5A), indicating that the high recombinogenic behavior of c-MYC in this system in hpr1Δ was due to both its high GC-content (as compared with LYS2) and length (as compared with Sµ350). Therefore, c-MYC behaves as a hotspot of recombination in hpr1Δ cells that is slightly increased by the action of AID.


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)

Analysis of recombinogenic potential of the c-MYC sequence.(A) AID-independent recombinogenic behavior of c-MYC in THO mutants. A 3.8-kb fragment of the human c-MYC, as well as the control 4.1-kb long LYS2 sequence, were cloned into the GFP-based direct-repeat recombination system. Details as in Figure 1. (B) Leu+ translocation frequencies in c-MYC-containing wild-type and hpr1Δ cells after HO-induced DSBs. Details as in Figure 2. (C) Analysis by PCR of CT breakpoints in hpr1Δ yeast at the c-MYC sequence. Breakpoints from cells with (grey) and without (black) AID-overexpression are indicated by arrows (top). The analysis of the breakpoint junctions is shown at the bottom. The c-MYC/HO cut site junctions from eight AID- and five AID+ independent Leu+ translocant strains are shown. The length of the c-MYC sequence within the reconstituted ACT1 intron in the translocant chromosome is indicated. Underlined nucleotide residues indicate microhomologies between the MYC and HO cut site sequences. The sequence downstream of the HO cleavage site is shown on top.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044682&req=5

pgen-1002009-g005: Analysis of recombinogenic potential of the c-MYC sequence.(A) AID-independent recombinogenic behavior of c-MYC in THO mutants. A 3.8-kb fragment of the human c-MYC, as well as the control 4.1-kb long LYS2 sequence, were cloned into the GFP-based direct-repeat recombination system. Details as in Figure 1. (B) Leu+ translocation frequencies in c-MYC-containing wild-type and hpr1Δ cells after HO-induced DSBs. Details as in Figure 2. (C) Analysis by PCR of CT breakpoints in hpr1Δ yeast at the c-MYC sequence. Breakpoints from cells with (grey) and without (black) AID-overexpression are indicated by arrows (top). The analysis of the breakpoint junctions is shown at the bottom. The c-MYC/HO cut site junctions from eight AID- and five AID+ independent Leu+ translocant strains are shown. The length of the c-MYC sequence within the reconstituted ACT1 intron in the translocant chromosome is indicated. Underlined nucleotide residues indicate microhomologies between the MYC and HO cut site sequences. The sequence downstream of the HO cleavage site is shown on top.
Mentions: Therefore, we cloned a 3.8-kb fragment of the human c-MYC gene that included the first exon and the first intron in our GFP direct-repeat recombination assay, as a functional and structural equivalent of a long S region. As can be seen in Figure 5A, whereas recombination was not affected by the presence of the MYC sequence in wild-type cells, regardless of AID expression, a strong increase was seen in hpr1Δ cells both without and with AID expression (24-fold and 32-fold increase with respect to the control wild-type, respectively; Figure 5A). To test whether these high recombination levels, notably higher than those obtained with the Sµ350 sequence (compare Figure 1 and Figure 5A), were not just due to the presence of a longer DNA sequence between the GFP repeats, we tested the effect of the long (4.1 kb) and low GC-rich (40%) sequence of the LYS2 gene. Recombination levels in this system containing the LYS2 sequence dropped markedly (Figure 5A), indicating that the high recombinogenic behavior of c-MYC in this system in hpr1Δ was due to both its high GC-content (as compared with LYS2) and length (as compared with Sµ350). Therefore, c-MYC behaves as a hotspot of recombination in hpr1Δ cells that is slightly increased by the action of AID.

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