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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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Effect of AID on recombination activity. (A) AID and Rad51 protein levels in the cell lines studied. (B) Factor of transcription effect of BJAB subclones without and with overexpression of HA-AID. (C) Comparison of endogenous and HA-tagged AID protein expression levels in Raji and BJAB cells transfected with the HA-AID- or NGFRt-expression vector. A minute lower migrating band seen in some experiments for endogenous AID likely represents a degradation product. (D) Recombination events in BJAB cells transfected with the reporter and the HA-AID- or NGFRt-expression vector. Completion of selection of cells bearing both the recombination reporter and the overexpression vector was achieved around day 16.
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Figure 3: Effect of AID on recombination activity. (A) AID and Rad51 protein levels in the cell lines studied. (B) Factor of transcription effect of BJAB subclones without and with overexpression of HA-AID. (C) Comparison of endogenous and HA-tagged AID protein expression levels in Raji and BJAB cells transfected with the HA-AID- or NGFRt-expression vector. A minute lower migrating band seen in some experiments for endogenous AID likely represents a degradation product. (D) Recombination events in BJAB cells transfected with the reporter and the HA-AID- or NGFRt-expression vector. Completion of selection of cells bearing both the recombination reporter and the overexpression vector was achieved around day 16.

Mentions: The Raji Burkitt lymphoma cell line is characterized by constitutive hypermutation activity, as determined by a hypermutation reporter in an identical vector context (18). The effect of transcription on recombination seen in these cells may be caused by AID, which is also active on transcribed genes. Indeed, one obvious difference between BJAB and Raji is their AID protein level (Figure 3A).Figure 3.


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)

Effect of AID on recombination activity. (A) AID and Rad51 protein levels in the cell lines studied. (B) Factor of transcription effect of BJAB subclones without and with overexpression of HA-AID. (C) Comparison of endogenous and HA-tagged AID protein expression levels in Raji and BJAB cells transfected with the HA-AID- or NGFRt-expression vector. A minute lower migrating band seen in some experiments for endogenous AID likely represents a degradation product. (D) Recombination events in BJAB cells transfected with the reporter and the HA-AID- or NGFRt-expression vector. Completion of selection of cells bearing both the recombination reporter and the overexpression vector was achieved around day 16.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Effect of AID on recombination activity. (A) AID and Rad51 protein levels in the cell lines studied. (B) Factor of transcription effect of BJAB subclones without and with overexpression of HA-AID. (C) Comparison of endogenous and HA-tagged AID protein expression levels in Raji and BJAB cells transfected with the HA-AID- or NGFRt-expression vector. A minute lower migrating band seen in some experiments for endogenous AID likely represents a degradation product. (D) Recombination events in BJAB cells transfected with the reporter and the HA-AID- or NGFRt-expression vector. Completion of selection of cells bearing both the recombination reporter and the overexpression vector was achieved around day 16.
Mentions: The Raji Burkitt lymphoma cell line is characterized by constitutive hypermutation activity, as determined by a hypermutation reporter in an identical vector context (18). The effect of transcription on recombination seen in these cells may be caused by AID, which is also active on transcribed genes. Indeed, one obvious difference between BJAB and Raji is their AID protein level (Figure 3A).Figure 3.

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