title: DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.
abstract: Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.
authors.affiliation: Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: Deniz
authors.initials: D
authors.lastname: Simsek
authors.forename: Erika
authors.initials: E
authors.lastname: Brunet
authors.forename: Sunnie Yan-Wai
authors.initials: SY
authors.lastname: Wong
authors.forename: Sachin
authors.initials: S
authors.lastname: Katyal
authors.forename: Yankun
authors.initials: Y
authors.lastname: Gao
authors.forename: Peter J
authors.initials: PJ
authors.lastname: McKinnon
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Lou
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: James
authors.initials: J
authors.lastname: Li
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Maria
authors.initials: M
authors.lastname: Jasin
mesh_terms: D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D000075223:Poly-ADP-Ribose Binding Proteins; D014162:Transfection; D014178:Translocation, Genetic; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; C000607894:Lig1 protein, mouse; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618818:Lig3 protein, mouse