TY - JOUR
T1 - Dissection of DNA double-strand-break repair using novel single-molecule forceps
AU - Wang, Jing L.
AU - Duboc, Camille
AU - Wu, Qian
AU - Ochi, Takashi
AU - Liang, Shikang
AU - Tsutakawa, Susan E.
AU - Lees-Miller, Susan P.
AU - Nadal, Marc
AU - Tainer, John A.
AU - Blundell, Tom L.
AU - Strick, Terence R.
N1 - Publisher Copyright:
© 2018 The Author(s).
PY - 2018/6/1
Y1 - 2018/6/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41594-018-0065-1
DO - 10.1038/s41594-018-0065-1
M3 - Article
C2 - 29786079
AN - SCOPUS:85047181534
SN - 1545-9993
VL - 25
SP - 482
EP - 487
JO - Nature Structural and Molecular Biology
JF - Nature Structural and Molecular Biology
IS - 6
ER -