title: Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.
abstract: Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Vandna
authors.initials: V
authors.lastname: Kukshal
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: In-Kwon
authors.initials: IK
authors.lastname: Kim
authors.affiliation: Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Hura
authors.affiliation: Department of Internal Medicine and University of New Mexico Cancer Center, University of New Mexico, Albuquerque, NM 87131, USA.
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.affiliation: Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA tome@biochem.wustl.edu.
authors.forename: Tom
authors.initials: T
authors.lastname: Ellenberger
mesh_terms: D004247:DNA / Q000378:metabolism*; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029867:Xenopus Proteins; D016335:Zinc Fingers
chemical_list: D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human