Abstract
In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg(64), Leu(65), and Leu(115)) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.
Highlights
In higher eukaryotes, DNA double-strand breaks4 are preferentially repaired by the nonhomologous end-joining (NHEJ) pathway composed of seven factors
Cernunnos depends on L4 for its inclusion within the X4-L4 ligation complex, which itself depends on X4 for the stability of L4 (8 –11)
We initially screened these mutations for their impact on V(D)J recombination, a DNA recombination process that critically relies on effective NHEJ and selected 10 of them for additional analyses
Summary
DNA double-strand breaks (dsb)4 are preferentially repaired by the nonhomologous end-joining (NHEJ) pathway composed of seven factors. To define better the Cernunnos structure-function relationship, we performed site-directed mutagenesis on 27 residues, located in various regions of the protein or corresponding to positions mutated in Cernunnos-deficient patients.
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