| Autor: |
Reid DA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;, Keegan S; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;, Leo-Macias A; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;, Watanabe G; Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and., Strande NT; Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599., Chang HH; Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and., Oksuz BA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;, Fenyo D; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;, Lieber MR; Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and., Ramsden DA; Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599., Rothenberg E; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; eli.rothenberg@nyumc.org. |
| Abstrakt: |
Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation. |
