Dissection of DNA double-strand-break repair using novel single-molecule forceps.

Autor: Wang JL; Institut Jacques Monod, Université Paris Diderot, CNRS, UMR 7592, Paris, France., Duboc C; Institut Jacques Monod, Université Paris Diderot, CNRS, UMR 7592, Paris, France., Wu Q; Department of Biochemistry, Cambridge University, Cambridge, UK., Ochi T; Department of Biochemistry, Cambridge University, Cambridge, UK.; MRC Laboratory of Molecular Biology, Cambridge, UK., Liang S; Department of Biochemistry, Cambridge University, Cambridge, UK., Tsutakawa SE; Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Lees-Miller SP; Department of Biochemistry and Molecular Biology, Arnie Charbonneau Cancer Institute, University of Calgary, Calgary, Alberta, Canada., Nadal M; Institut Jacques Monod, Université Paris Diderot, CNRS, UMR 7592, Paris, France., Tainer JA; Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.; Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA., Blundell TL; Department of Biochemistry, Cambridge University, Cambridge, UK., Strick TR; Institut Jacques Monod, Université Paris Diderot, CNRS, UMR 7592, Paris, France. strick@ens.fr.; Ecole Normale Supérieure, PSL Research University, CNRS, INSERM, Institute of Biology (IBENS), Paris, France. strick@ens.fr.; Programme Equipes Labellisées, Ligue Contre le Cancer, Paris, France. strick@ens.fr.
Jazyk: angličtina
Zdroj: Nature structural & molecular biology [Nat Struct Mol Biol] 2018 Jun; Vol. 25 (6), pp. 482-487. Date of Electronic Publication: 2018 May 21.
DOI: 10.1038/s41594-018-0065-1
Abstrakt: Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.
Databáze: MEDLINE
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