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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.