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Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells.

Han L, Yu K - J. Exp. Med. (2008)

Bottom Line: By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR.However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4- cells.The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4- cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

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Related in: MedlinePlus

Sμ–Sα junction sequences. (A) Percentage of switch junctions with the indicated length of microhomology (excluding nucleotide additions). (B and C) Alignment of switch junctions with germline sequences. Germline Sμ (black) and Sα (gray) sequences are listed on the top and bottom, respectively, of each junction sequence (blue). Microhomologies (boxes) are identified as the largest perfect matches to the germline sequences. Nucleotide additions are underlined. Long vertical lines indicate direct joins. Small vertical lines indicate identity between the junction and germline sequences. Several junctions containing inverted Sμ are indicated by arrows.
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fig5: Sμ–Sα junction sequences. (A) Percentage of switch junctions with the indicated length of microhomology (excluding nucleotide additions). (B and C) Alignment of switch junctions with germline sequences. Germline Sμ (black) and Sα (gray) sequences are listed on the top and bottom, respectively, of each junction sequence (blue). Microhomologies (boxes) are identified as the largest perfect matches to the germline sequences. Nucleotide additions are underlined. Long vertical lines indicate direct joins. Small vertical lines indicate identity between the junction and germline sequences. Several junctions containing inverted Sμ are indicated by arrows.

Mentions: Numerous studies have shown increased use of microhomology at the junctions in NHEJ-deficient cells (14, 21, 22). To determine whether switch junctions in Lig4- cells are microhomology mediated, we cloned and sequenced Sμ–Sα junctions from stimulated Lig4-heterozygous and Lig4- cells. Because Sα is highly homologous to Sμ, we chose to PCR amplify switch junctions from individual cell clones. Cloning individual switch junctions instead of amplifying from a pool of cells helps to eliminate false junctions produced by template switching of stalled PCR intermediates, as well as the bias against large amplicons. In addition, the distinctive size of each PCR product was used to reference the authenticity of the sequenced junction. We found that 27% of the junctions from Lig4-heterozygous cells were direct joins, but there were none for the Lig4- cells (Fig. 5). The difference is statistically significant (7 out of 26 vs. 0 out of 23; two-tail p-value of 0.01 by Fisher's exact test) and consistent with what was observed for the Sμ–Sγ switch junctions in XRCC4-deficient B cells (14). The distribution of recombination break points is indistinguishable between the two genotypes (unpublished data).


Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells.

Han L, Yu K - J. Exp. Med. (2008)

Sμ–Sα junction sequences. (A) Percentage of switch junctions with the indicated length of microhomology (excluding nucleotide additions). (B and C) Alignment of switch junctions with germline sequences. Germline Sμ (black) and Sα (gray) sequences are listed on the top and bottom, respectively, of each junction sequence (blue). Microhomologies (boxes) are identified as the largest perfect matches to the germline sequences. Nucleotide additions are underlined. Long vertical lines indicate direct joins. Small vertical lines indicate identity between the junction and germline sequences. Several junctions containing inverted Sμ are indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2585838&req=5

fig5: Sμ–Sα junction sequences. (A) Percentage of switch junctions with the indicated length of microhomology (excluding nucleotide additions). (B and C) Alignment of switch junctions with germline sequences. Germline Sμ (black) and Sα (gray) sequences are listed on the top and bottom, respectively, of each junction sequence (blue). Microhomologies (boxes) are identified as the largest perfect matches to the germline sequences. Nucleotide additions are underlined. Long vertical lines indicate direct joins. Small vertical lines indicate identity between the junction and germline sequences. Several junctions containing inverted Sμ are indicated by arrows.
Mentions: Numerous studies have shown increased use of microhomology at the junctions in NHEJ-deficient cells (14, 21, 22). To determine whether switch junctions in Lig4- cells are microhomology mediated, we cloned and sequenced Sμ–Sα junctions from stimulated Lig4-heterozygous and Lig4- cells. Because Sα is highly homologous to Sμ, we chose to PCR amplify switch junctions from individual cell clones. Cloning individual switch junctions instead of amplifying from a pool of cells helps to eliminate false junctions produced by template switching of stalled PCR intermediates, as well as the bias against large amplicons. In addition, the distinctive size of each PCR product was used to reference the authenticity of the sequenced junction. We found that 27% of the junctions from Lig4-heterozygous cells were direct joins, but there were none for the Lig4- cells (Fig. 5). The difference is statistically significant (7 out of 26 vs. 0 out of 23; two-tail p-value of 0.01 by Fisher's exact test) and consistent with what was observed for the Sμ–Sγ switch junctions in XRCC4-deficient B cells (14). The distribution of recombination break points is indistinguishable between the two genotypes (unpublished data).

Bottom Line: By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR.However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4- cells.The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.

ABSTRACT
Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4- cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

Show MeSH
Related in: MedlinePlus