Formation and resolution of double-strand break intermediates in V(D)J rearrangement.
A recently described pre-B cell line can be induced at high temperature to actively rearrange its immunoglobulin light-chain loci. We used this cell line to determine the fate of double-strand breaks generated by V(D)J rearrangement. After induction, 30%-40% of K loci had broken JK1 signal ends. JK1-coding ends were detectable, but 10- to 100-fold less frequent. Both covalently closed (hairpin) and open, blunt, processed coding ends were observed. Coding junctions involving JK1 accumulated with similar kinetics as JK1 signal ends, arguing that coding ends can be resolved quickly and efficiently to coding junctions, whereas signal ends remain mostly unjoined. Signal ends are then joined rapidly when cells are returned to the low temperature. These results support the model that broken signal ends and hairpin coding ends are authentic intermediates in V(D)J recombination. It appears that hairpin coding ends are rapidly opened, processed, and resolved to coding junctions, whereas joining of signal ends is clearly uncoupled from the joining of coding ends and can be much slower. Efficient formation of signal junctions may require cell cycle progression, or down-regulation of the recombination machinery.
Ramsden
D A
DA
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
Gellert
M
M
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006358:Hot Temperature; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014170:Transformation, Genetic / Q000235:genetics; D045567:VDJ Recombinases
D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases