Metabolism of recombination coding ends in scid cells.
V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3' overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.
Brown
M L
ML
Department of Microbiology, Arizona State University, Tempe, AZ 85287, USA.
Chang
Y
Y
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed; D003062:Codon / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015322:Gene Rearrangement, B-Lymphocyte / Q000276:immunology*; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007700:Kinetics; D000076228:MRE11 Homologue Protein; D051379:Mice; D016513:Mice, SCID; D008822:Mice, Transgenic; D011995:Recombination, Genetic / Q000276:immunology*; D013696:Temperature; D013997:Time Factors; D019895:Transposases / Q000096:biosynthesis / Q000235:genetics
D003062:Codon; D004268:DNA-Binding Proteins; D007144:Immunoglobulin J-Chains; D007145:Immunoglobulin kappa-Chains; C512689:Mre11a protein, mouse; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; D019895:Transposases; D000076228:MRE11 Homologue Protein; D045643:DNA Repair Enzymes