BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair
BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair
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DOI：
10.1101/gad.2003811
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发表时间：
2011-02-15
影响因子：
10.5
通讯作者：
Kowalczykowski, Stephen C.
中科院分区：
文献类型：
--
作者：
Nimonkar, Amitabh V.;Genschel, Jochen;Kowalczykowski, Stephen C.
DsDNA断裂的修复需要加工以产生3‘端终止的单链DNA。我们使用纯化的人类蛋白质：Bloom Helicase(BLM)；DNA2解旋酶/核酸酶；外切核酸酶1(EXO1)；由Mre11、Rad50和NBS1(MRN)组成的复合体；以及复制蛋白A(RPA)，以生物化学的方式重建DNA末端切除。切除通过两种途径进行。在一个过程中，BLM和DNA2在物理上和特异性地相互作用，在一个依赖于ATP的过程中切除DNA，这需要BLM解旋酶和DNA2核酸酶的功能。RPA对于BLM解开DNA和DNA2加强5‘-gt；3’切除极性都是必不可少的。MRN通过将BLM招募到最后来加快处理速度。在另一种情况下，EXO1切除DNA，并受到博莱曼、MRN和RPA的刺激。BLM增加EXO1对END的亲和力，MRN招募并增强EXO1的加工能力。我们的结果建立了在人类细胞中启动重组DNA修复的两个核心机制。
Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5' -> 3' resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.