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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 the proportion of conservative recombination in human B cells. (A) Schematic structure of the previously used reporter for all recombination pathways (tHR) and the reporter specific for conservative recombination (cHR). (B) Flow cytometry dot plots for frequencies of GFP+ cells in the Burkitt and Burkitt like lymphoma cell lines Raji, BJAB and DG75 and the lymphoblastoid cell line 721 for the reporter detecting all recombination events (tHR, upper row) and the reporter specific for conservative recombination (cHR, bottom row). (C) Percentage of conservative recombination in the cell lines analysed in B. Absolute numbers are listed in Table 1. (D) Rad51 overexpression does not rescue conservative HR in Raji cells. A clone carrying the inducible Rad51 overexpression vector was transfected with the tHR and cHR reporters, and upon completion of selection (day 20), Rad51 was induced in one aliquot of the culture by doxycycline addition. (E) Successful Rad51 induction in the analyses shown in (D) was assessed by western blot.
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Figure 5: Differences in the proportion of conservative recombination in human B cells. (A) Schematic structure of the previously used reporter for all recombination pathways (tHR) and the reporter specific for conservative recombination (cHR). (B) Flow cytometry dot plots for frequencies of GFP+ cells in the Burkitt and Burkitt like lymphoma cell lines Raji, BJAB and DG75 and the lymphoblastoid cell line 721 for the reporter detecting all recombination events (tHR, upper row) and the reporter specific for conservative recombination (cHR, bottom row). (C) Percentage of conservative recombination in the cell lines analysed in B. Absolute numbers are listed in Table 1. (D) Rad51 overexpression does not rescue conservative HR in Raji cells. A clone carrying the inducible Rad51 overexpression vector was transfected with the tHR and cHR reporters, and upon completion of selection (day 20), Rad51 was induced in one aliquot of the culture by doxycycline addition. (E) Successful Rad51 induction in the analyses shown in (D) was assessed by western blot.

Mentions: To study the ratio of non-conservative and conservative HR in these cells in more detail, we additionally applied a reporter specific for conservative recombination (Figure 5A). This specificity can be achieved by locating the donor GFP gene upstream of the acceptor gene or by inactivation of the donor GFP gene by deletion of both the 5′- and the 3′-end of the coding sequence. Due to these modifications, non-conservative recombination cannot restore a functional GFP gene. We chose the latter approach to ensure better comparability of the two reporters.Figure 5.


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 the proportion of conservative recombination in human B cells. (A) Schematic structure of the previously used reporter for all recombination pathways (tHR) and the reporter specific for conservative recombination (cHR). (B) Flow cytometry dot plots for frequencies of GFP+ cells in the Burkitt and Burkitt like lymphoma cell lines Raji, BJAB and DG75 and the lymphoblastoid cell line 721 for the reporter detecting all recombination events (tHR, upper row) and the reporter specific for conservative recombination (cHR, bottom row). (C) Percentage of conservative recombination in the cell lines analysed in B. Absolute numbers are listed in Table 1. (D) Rad51 overexpression does not rescue conservative HR in Raji cells. A clone carrying the inducible Rad51 overexpression vector was transfected with the tHR and cHR reporters, and upon completion of selection (day 20), Rad51 was induced in one aliquot of the culture by doxycycline addition. (E) Successful Rad51 induction in the analyses shown in (D) was assessed by western blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553578&req=5

Figure 5: Differences in the proportion of conservative recombination in human B cells. (A) Schematic structure of the previously used reporter for all recombination pathways (tHR) and the reporter specific for conservative recombination (cHR). (B) Flow cytometry dot plots for frequencies of GFP+ cells in the Burkitt and Burkitt like lymphoma cell lines Raji, BJAB and DG75 and the lymphoblastoid cell line 721 for the reporter detecting all recombination events (tHR, upper row) and the reporter specific for conservative recombination (cHR, bottom row). (C) Percentage of conservative recombination in the cell lines analysed in B. Absolute numbers are listed in Table 1. (D) Rad51 overexpression does not rescue conservative HR in Raji cells. A clone carrying the inducible Rad51 overexpression vector was transfected with the tHR and cHR reporters, and upon completion of selection (day 20), Rad51 was induced in one aliquot of the culture by doxycycline addition. (E) Successful Rad51 induction in the analyses shown in (D) was assessed by western blot.
Mentions: To study the ratio of non-conservative and conservative HR in these cells in more detail, we additionally applied a reporter specific for conservative recombination (Figure 5A). This specificity can be achieved by locating the donor GFP gene upstream of the acceptor gene or by inactivation of the donor GFP gene by deletion of both the 5′- and the 3′-end of the coding sequence. Due to these modifications, non-conservative recombination cannot restore a functional GFP gene. We chose the latter approach to ensure better comparability of the two reporters.Figure 5.

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