title: Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells.
abstract: V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. Thus, recombination ends may either be retained in a complex throughout the recombination process or left as unprotected free ends after cleavage, a condition that may permit an alternative, non-classical NHEJ end joining pathway. To directly test these scenarios on recombination induced chromosomal breaks, we have developed a recombination end protection assay to monitor the accessibility of recombination ends to exonuclease-V in intact nuclei. We demonstrate that these ends are well protected in the nuclei of wild-type cells, suggesting a seamless cleavage-joining reaction. However, divergent end protection of coding versus signal ends was found in cells derived from severe combined immunodeficient (scid) mice that are defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While signal ends are resistant, opened coding ends are susceptible to enzymatic modification. Our data suggests a role of DNA-PKcs in protecting chromosomal coding ends. Furthermore, using recombination inducible scid cell lines, we demonstrate that conditional protection of coding ends is inversely correlated with the level of their resolution, i.e., the greater the accessibility of the coding ends, the higher level of coding joints formed. Taken together, our findings provide important insights into the resolution of recombination ends by error-prone alternative NHEJ pathways.
authors.affiliation: Molecular and Cellular Biology Program, School of Life Sciences, Center for Infectious Diseases and Vaccinology, The Biodesign Institute at Arizona State University, BDA220, 1001 S. McAllister Avenue, Tempe, AZ 85287-5501, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Franco
authors.forename: Yung
authors.initials: Y
authors.lastname: Chang
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019457:Chromosome Breakage; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D015321:Gene Rearrangement; D005838:Genotype; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D016513:Mice, SCID; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013696:Temperature; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D045567:VDJ Recombinases / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C499308:Rag2 protein, mouse; D051747:DNA-Activated Protein Kinase; D045567:VDJ Recombinases