Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links
Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links
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DOI：
10.1074/jbc.m112.419358
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发表时间：
2013-02-15
影响因子：
4.8
通讯作者：
Ide, Hiroshi
中科院分区：
文献类型：
--
作者：
Nakano, Toshiaki;Miyamoto-Matsubara, Mayumi;Ide, Hiroshi
当细胞暴露于各种DNA损伤剂时，形成DNA-蛋白质交联（DPC）。由于DPC非常大，DPC赋予的空间位阻可能会影响DNA交易的许多方面。在DNA复制中，DPC首先被在复制体头部移动的复制解旋酶遇到。然而，很少有人知道如何复制解旋酶响应共价固定的蛋白质路障。在本研究中，我们阐明了DPC的DNA解旋反应的六聚体复制解旋酶在体外使用定义的DPC底物的效果。DPC的易位链，但不对nontranslocating链阻碍的解旋酶，包括噬菌体T7基因4蛋白，猿猴病毒40大T抗原，大肠杆菌DnaB蛋白，和人类微小染色体维护Mcm 467亚复合物的进展。该障碍随交联蛋白的大小而变化，清除的阈值大小为5.0-14.1 kDa。这些结果表明，动态易位的六聚体环解旋酶的中央通道可以容纳只有小的蛋白质和所有的解旋酶测试使用的空间排阻机制解开双链体DNA。这些结果进一步表明，DPC的易位和nontranslocating链构成解旋酶和聚合酶块，分别。解旋酶停止DPC有有限的稳定性和解离的DNA的半衰期为15-36分钟。结果的影响进行了讨论，在不同的稳定性的复制体，遇到紧密的，但可逆的DNA-蛋白质复合物和不可逆的DPC路障。
DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0-14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15-36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.