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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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Differences in HR activity in human B cells. (A) The GFP-based recombination cassette and the two potential recombination products. The acceptor GFP contains a frameshift insertion, and the donor GFP is inactivated by an N-terminal deletion (striped boxes). CMV-P, CMV promoter; Ig enhancer elements: E3, 3′ enhancer; Ei, intron enhancer; MAR, matrix attachment region. Epstein-Barr-virus elements: replication origin OriP and the EBV nuclear antigen (EBNA) 1 gene. (B) Detection of GFP+ cells over time in Raji cells transfected with the recombination reporter (upper plots) or a reporter lacking the donor GFP (lower plots). (C) Frequencies of GFP+ cells over time in the Burkitt lymphoma line Raji, the Burkitt-like line BJAB, the two human lymphoblastoid cell lines 721 and EREB2-5 and the T cell line Jurkat. B cells were transfected with the recombination reporter containing Ig enhancers and Jurkat with a construct lacking the enhancers. Selection for vector bearing cells was completed around day 14. Data are representative of ≥5 (Raji, BJAB, 721) or 2 (Jurkat, EREB2-5) experiments. (D) Frequencies of GFP restoring events per vector copy over time in the respective cell lines.
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Figure 1: Differences in HR activity in human B cells. (A) The GFP-based recombination cassette and the two potential recombination products. The acceptor GFP contains a frameshift insertion, and the donor GFP is inactivated by an N-terminal deletion (striped boxes). CMV-P, CMV promoter; Ig enhancer elements: E3, 3′ enhancer; Ei, intron enhancer; MAR, matrix attachment region. Epstein-Barr-virus elements: replication origin OriP and the EBV nuclear antigen (EBNA) 1 gene. (B) Detection of GFP+ cells over time in Raji cells transfected with the recombination reporter (upper plots) or a reporter lacking the donor GFP (lower plots). (C) Frequencies of GFP+ cells over time in the Burkitt lymphoma line Raji, the Burkitt-like line BJAB, the two human lymphoblastoid cell lines 721 and EREB2-5 and the T cell line Jurkat. B cells were transfected with the recombination reporter containing Ig enhancers and Jurkat with a construct lacking the enhancers. Selection for vector bearing cells was completed around day 14. Data are representative of ≥5 (Raji, BJAB, 721) or 2 (Jurkat, EREB2-5) experiments. (D) Frequencies of GFP restoring events per vector copy over time in the respective cell lines.

Mentions: To study HR in human B cells, we used an episomally replicating EBV-based vector carrying a CMV promoter-driven direct repeat of two defective GFP genes. The acceptor GFP gene is inactivated by a frameshift insertion and can be reconstituted to a functional GFP by HR with the donor GFP gene, of which the 5′ coding sequence is deleted (Figure 1A) (17). The reporter is able to differentiate between conservative and non-conservative recombination pathways (Figure 1A), each of which may be based on multiple molecular mechanisms [see (3–6) and Discussion section for details].Figure 1.


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)

Differences in HR activity in human B cells. (A) The GFP-based recombination cassette and the two potential recombination products. The acceptor GFP contains a frameshift insertion, and the donor GFP is inactivated by an N-terminal deletion (striped boxes). CMV-P, CMV promoter; Ig enhancer elements: E3, 3′ enhancer; Ei, intron enhancer; MAR, matrix attachment region. Epstein-Barr-virus elements: replication origin OriP and the EBV nuclear antigen (EBNA) 1 gene. (B) Detection of GFP+ cells over time in Raji cells transfected with the recombination reporter (upper plots) or a reporter lacking the donor GFP (lower plots). (C) Frequencies of GFP+ cells over time in the Burkitt lymphoma line Raji, the Burkitt-like line BJAB, the two human lymphoblastoid cell lines 721 and EREB2-5 and the T cell line Jurkat. B cells were transfected with the recombination reporter containing Ig enhancers and Jurkat with a construct lacking the enhancers. Selection for vector bearing cells was completed around day 14. Data are representative of ≥5 (Raji, BJAB, 721) or 2 (Jurkat, EREB2-5) experiments. (D) Frequencies of GFP restoring events per vector copy over time in the respective cell lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Differences in HR activity in human B cells. (A) The GFP-based recombination cassette and the two potential recombination products. The acceptor GFP contains a frameshift insertion, and the donor GFP is inactivated by an N-terminal deletion (striped boxes). CMV-P, CMV promoter; Ig enhancer elements: E3, 3′ enhancer; Ei, intron enhancer; MAR, matrix attachment region. Epstein-Barr-virus elements: replication origin OriP and the EBV nuclear antigen (EBNA) 1 gene. (B) Detection of GFP+ cells over time in Raji cells transfected with the recombination reporter (upper plots) or a reporter lacking the donor GFP (lower plots). (C) Frequencies of GFP+ cells over time in the Burkitt lymphoma line Raji, the Burkitt-like line BJAB, the two human lymphoblastoid cell lines 721 and EREB2-5 and the T cell line Jurkat. B cells were transfected with the recombination reporter containing Ig enhancers and Jurkat with a construct lacking the enhancers. Selection for vector bearing cells was completed around day 14. Data are representative of ≥5 (Raji, BJAB, 721) or 2 (Jurkat, EREB2-5) experiments. (D) Frequencies of GFP restoring events per vector copy over time in the respective cell lines.
Mentions: To study HR in human B cells, we used an episomally replicating EBV-based vector carrying a CMV promoter-driven direct repeat of two defective GFP genes. The acceptor GFP gene is inactivated by a frameshift insertion and can be reconstituted to a functional GFP by HR with the donor GFP gene, of which the 5′ coding sequence is deleted (Figure 1A) (17). The reporter is able to differentiate between conservative and non-conservative recombination pathways (Figure 1A), each of which may be based on multiple molecular mechanisms [see (3–6) and Discussion section for details].Figure 1.

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