RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions.
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 lambdaR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-lambdaR, showed that RAD51D and XRCC2 localize to the diversifying lambdaR 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.


CONCLUSIONS
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.
Ordinario
Ellen C
EC
Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195-7650, USA. ecordinario@lbl.gov
Yabuki
Munehisa
M
Handa
Priya
P
Cummings
W Jason
WJ
Maizels
Nancy
N
D000483:Alleles; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D003564:Cytidine Deaminase / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D005785:Gene Conversion; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051135:Rad51 Recombinase / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004268:DNA-Binding Proteins; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051135:Rad51 Recombinase; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase