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Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

Mierau M, Drexler GA, Kutzera A, Braunschmidt K, Ellwart J, Eckardt-Schupp F, Fritz E, Bachl J, Jungnickel B - Nucleic Acids Res. (2008)

Bottom Line: The molecular basis for the differential use of these two pathways in different species is unclear.Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID.Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich, National Research Center for Environmental Health, D-81377 Munich.

ABSTRACT
During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

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Preferential non-conservative recombination in human B cells. (A) Structural and sequence analysis of recovered reporter plasmids from Raji cells (after 35 days of culture) showing a GFP restoring recombination event (R; thick bar) or destructive deletions (D1-5; thin bars). Gaps in the sequence information display junctions with micro-homologies (grey) or insertions (bold). (B) Analysis for GFP expression capacity of two recovered reporter plasmids (R and D3) by retransfection into Raji cells. GFP indicates the control with a vector containing the functional GFP gene. (C) Schematic view of the PCR/restriction approach for analysis of recombination products. Primers for the respective PCRs (arrowheads) and the Bst1107I restriction site used for discrimination of repaired GFP genes are shown. (D) PCR/restriction/Southern blot analysis of DNA from sorted GFP+ cells from Raji and BJAB cells. Two independent experiments are shown for Raji. The transfected vector was used as a negative control. Full-length GFP was used as probe. (E) PCR/restriction/Southern blot analysis of chromosomally integrated and episomal reporters in Raji and BJAB cells analysed as in D. (F) Comparison of the GFP fluorescence intensity of the two possible recombination products (see C) in BJAB and Raji cells. Black line: non-conservative recombination product; grey: conservative recombination product. Corresponding mean fluorescence intensities (MFI) are listed.
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Figure 4: Preferential non-conservative recombination in human B cells. (A) Structural and sequence analysis of recovered reporter plasmids from Raji cells (after 35 days of culture) showing a GFP restoring recombination event (R; thick bar) or destructive deletions (D1-5; thin bars). Gaps in the sequence information display junctions with micro-homologies (grey) or insertions (bold). (B) Analysis for GFP expression capacity of two recovered reporter plasmids (R and D3) by retransfection into Raji cells. GFP indicates the control with a vector containing the functional GFP gene. (C) Schematic view of the PCR/restriction approach for analysis of recombination products. Primers for the respective PCRs (arrowheads) and the Bst1107I restriction site used for discrimination of repaired GFP genes are shown. (D) PCR/restriction/Southern blot analysis of DNA from sorted GFP+ cells from Raji and BJAB cells. Two independent experiments are shown for Raji. The transfected vector was used as a negative control. Full-length GFP was used as probe. (E) PCR/restriction/Southern blot analysis of chromosomally integrated and episomal reporters in Raji and BJAB cells analysed as in D. (F) Comparison of the GFP fluorescence intensity of the two possible recombination products (see C) in BJAB and Raji cells. Black line: non-conservative recombination product; grey: conservative recombination product. Corresponding mean fluorescence intensities (MFI) are listed.

Mentions: One of the six altered plasmids (R) showed a repair event where the two GFP genes are fused (Figure 4A), restoring the functionality of the GFP gene as demonstrated in Figure 4B. In our reporter system, this kind of recombination product may arise either from rare gene conversion with a crossover event, or alternatively from pathways that are grouped as non-conservative HR and are defined by such net loss of DNA.Figure 4.


Non-conservative homologous recombination in human B lymphocytes is promoted by activation-induced cytidine deaminase and transcription.

Mierau M, Drexler GA, Kutzera A, Braunschmidt K, Ellwart J, Eckardt-Schupp F, Fritz E, Bachl J, Jungnickel B - Nucleic Acids Res. (2008)

Preferential non-conservative recombination in human B cells. (A) Structural and sequence analysis of recovered reporter plasmids from Raji cells (after 35 days of culture) showing a GFP restoring recombination event (R; thick bar) or destructive deletions (D1-5; thin bars). Gaps in the sequence information display junctions with micro-homologies (grey) or insertions (bold). (B) Analysis for GFP expression capacity of two recovered reporter plasmids (R and D3) by retransfection into Raji cells. GFP indicates the control with a vector containing the functional GFP gene. (C) Schematic view of the PCR/restriction approach for analysis of recombination products. Primers for the respective PCRs (arrowheads) and the Bst1107I restriction site used for discrimination of repaired GFP genes are shown. (D) PCR/restriction/Southern blot analysis of DNA from sorted GFP+ cells from Raji and BJAB cells. Two independent experiments are shown for Raji. The transfected vector was used as a negative control. Full-length GFP was used as probe. (E) PCR/restriction/Southern blot analysis of chromosomally integrated and episomal reporters in Raji and BJAB cells analysed as in D. (F) Comparison of the GFP fluorescence intensity of the two possible recombination products (see C) in BJAB and Raji cells. Black line: non-conservative recombination product; grey: conservative recombination product. Corresponding mean fluorescence intensities (MFI) are listed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553578&req=5

Figure 4: Preferential non-conservative recombination in human B cells. (A) Structural and sequence analysis of recovered reporter plasmids from Raji cells (after 35 days of culture) showing a GFP restoring recombination event (R; thick bar) or destructive deletions (D1-5; thin bars). Gaps in the sequence information display junctions with micro-homologies (grey) or insertions (bold). (B) Analysis for GFP expression capacity of two recovered reporter plasmids (R and D3) by retransfection into Raji cells. GFP indicates the control with a vector containing the functional GFP gene. (C) Schematic view of the PCR/restriction approach for analysis of recombination products. Primers for the respective PCRs (arrowheads) and the Bst1107I restriction site used for discrimination of repaired GFP genes are shown. (D) PCR/restriction/Southern blot analysis of DNA from sorted GFP+ cells from Raji and BJAB cells. Two independent experiments are shown for Raji. The transfected vector was used as a negative control. Full-length GFP was used as probe. (E) PCR/restriction/Southern blot analysis of chromosomally integrated and episomal reporters in Raji and BJAB cells analysed as in D. (F) Comparison of the GFP fluorescence intensity of the two possible recombination products (see C) in BJAB and Raji cells. Black line: non-conservative recombination product; grey: conservative recombination product. Corresponding mean fluorescence intensities (MFI) are listed.
Mentions: One of the six altered plasmids (R) showed a repair event where the two GFP genes are fused (Figure 4A), restoring the functionality of the GFP gene as demonstrated in Figure 4B. In our reporter system, this kind of recombination product may arise either from rare gene conversion with a crossover event, or alternatively from pathways that are grouped as non-conservative HR and are defined by such net loss of DNA.Figure 4.

Bottom Line: The molecular basis for the differential use of these two pathways in different species is unclear.Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID.Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich, National Research Center for Environmental Health, D-81377 Munich.

ABSTRACT
During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.

Show MeSH
Related in: MedlinePlus