Research article

RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions

Ellen C Ordinario^1,3 , Munehisa Yabuki² , Priya Handa² , W Jason Cummings² and Nancy Maizels^1,2

¹Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7650 USA

²Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195-7650, USA

³Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA, USA

author email corresponding author email

BMC Molecular Biology 2009, 10:98doi:10.1186/1471-2199-10-98

Published:	28 October 2009

Abstract

Background

Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells.

Results

We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin λ_R light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-λ_R, showed that RAD51D and XRCC2 localize to the diversifying λ_R gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants.

Conclusion

These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.