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DNA Repair (Amst). 2014 Mar;15:39-53. doi: 10.1016/j.dnarep.2013.12.006. Epub 2014 Jan 21.

A role for XLF in DNA repair and recombination in human somatic cells.

Author information

1
Departments of Pharmacology and Radiation Oncology, Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States. Electronic address: Farjana.Fattah@utsouthwestern.edu.
2
Department of Pediatrics, Section of Cardiology, University of Chicago, 900 East 57th Street, KCBD Room 5240, Chicago, IL 60637, United States. Electronic address: jkweon@uchicago.edu.
3
Cancer Diagnostics Service, Quest Diagnostics Nichols Institute, Chantilly, VA 20151, United States. Electronic address: yongbao.x.wang@questdiagnostics.com.
4
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, United States.
5
University of ND School of Medicine, 501 Columbia Road, Grand Forks, ND 58203, United States. Electronic address: nlicht046@gmail.com.
6
University of Wisconsin School of Medicine and Public Health, Health Sciences Learning Center, 750 Highland Ave., Madison, WI 53705, United States. Electronic address: nweisensel@wisc.edu.
7
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, United States. Electronic address: hendr064@umn.edu.

Abstract

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.

PMID:
24461734
PMCID:
PMC3948327
DOI:
10.1016/j.dnarep.2013.12.006
[Indexed for MEDLINE]
Free PMC Article

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