The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.

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doi.org/AID-IMMU701%3E3.0.CO;2-T, hdl.handle.net/1765/3197
European Journal of Immunology
Erasmus MC: University Medical Center Rotterdam

Verkaik, N.S, Esveldt-van Lange, R, van Heemst, D, Brüggenwirth, H.T, Hoeijmakers, J.H.J, Zdzienicka, M.Z, & van Gent, D.C. (2002). Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells. European Journal of Immunology, 32(3), 701–709. doi:AID-IMMU701%3E3.0.CO;2-T