RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex
RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex
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DOI：
10.1093/carcin/bgu137
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发表时间：
2014-11-01
期刊：
影响因子：
4.7
通讯作者：
Bohr, Vilhelm A.
中科院分区：
文献类型：
--
作者：
Shamanna, Raghavendra A.;Singh, Dharmendra Kumar;Bohr, Vilhelm A.
RECQL 4是RecQ解旋酶家族的成员，是DNA代谢的多功能参与者。RECQL 4蛋白参与细胞核和细胞质中的几种功能，人类RECQL 4的突变与三种遗传疾病相关：Rothmund-Thomson综合征、RAPADILINO综合征和Baller-Cerold综合征。我们以前报道RECQL 4被招募到激光诱导的DNA双链断裂（DSB）。在这里，我们已经表征了RECQL 4在DSB修复的非同源末端连接（NHEJ）途径中的功能作用。在取决于DNA依赖性蛋白激酶（DNA-PK）活性的体外NHEJ测定中，RECQL 4敲除细胞的提取物在具有粘性和非粘性末端的DNA底物上显示出降低的末端连接活性。RECQL 4的消耗也降低了CFP报告质粒在体内的末端连接活性。RECQL 4的敲除增加了细胞对γ-辐射的敏感性，并导致辐射后53 BP 1灶的积累，表明DSB加工中的缺陷。我们发现RECQL 4通过其N-末端结构域与DNA-PK复合物的一部分Ku 70/Ku 80异二聚体相互作用。此外，RECQL 4刺激Ku 70/Ku 80与平端DNA底物的高阶DNA结合。综上所述，这些结果表明RECQL 4通过与Ku 70/Ku 80复合物的功能相互作用参与DSB修复的NHEJ途径。这是第一项研究，提供了在体外和体内的RecQ解旋酶在NHEJ中的作用的证据。
RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Cerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a CFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to gamma-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.