title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation.
abstract: The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4 in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.
authors.affiliation: College of Veterinary Medicine and Departments of Microbiology & Molecular Genetics and Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Sunetra
authors.initials: S
authors.lastname: Roy
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS, UMR7258, Marseille, France INSERM, U1068, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France.
authors.forename: Abinadabe J
authors.initials: AJ
authors.lastname: de Melo
authors.affiliation: College of Veterinary Medicine and Departments of Microbiology & Molecular Genetics and Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Yao
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS, UMR7258, Marseille, France INSERM, U1068, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France.
authors.forename: Satish K
authors.initials: SK
authors.lastname: Tadi
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS, UMR7258, Marseille, France INSERM, U1068, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Négrel
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Hendrickson
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS, UMR7258, Marseille, France INSERM, U1068, Marseille, France Institut Paoli-Calmettes, Marseille, France Aix-Marseille Université, Marseille, France.
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.affiliation: College of Veterinary Medicine and Departments of Microbiology & Molecular Genetics and Pathobiology & Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA kmeek@msu.edu.
authors.forename: Katheryn
authors.initials: K
authors.lastname: Meek
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000096:biosynthesis / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D017422:Sequence Analysis, DNA; D060152:V(D)J Recombination / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C508551:NHEJ1 protein, human; C000595075:PAXX protein, human; C101467:XRCC4 protein, human; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP