Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor.
Bibliographical noteFunding Information:
The authors are indebted to Drs. S. Jackson (Cambridge University, UK), V. Gorbunova (University of Rochester, NY), G. Iliakis (University of Duisburg-Essen Medical School, Germany), M. Jasin (Sloan Kettering, NY), J. Stark (City of Hope, CA) and D. van Gent (Erasmus University, Netherlands) and members of their laboratories who were extremely generous with their reagents and advice. We thank Dr. A.-K. Bielinsky for her careful reading of the manuscript and helpful comments. We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the Masonic Cancer Center, a comprehensive cancer center designated by the National Cancer Institute, supported in part by P30 CA77598. These studies were supported in part by a National Institutes of Health grant GM088351 and a National Cancer Institute grant CA154461 to EAH.