Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction.
Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction.
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在没有 ATP 的情况下，NS3 解旋酶结构域通过与单链/双链连接处的特异性相互作用来解链双链 DNA。
DOI：
10.1021/acs.biochem.5b00214
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发表时间：
2015
期刊：
影响因子：
2.9
通讯作者：
Raney,KevinD
中科院分区：
文献类型：
--
作者：
Reynolds,KimberlyA;Cameron,CraigE;Raney,KevinD
Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. Nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3′-to-5′ direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, the NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3′-tail (i.e., 3′-to-5′ directionality). Blunt-ended duplexes and 5′-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nucleotides2s–1. Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with an excess protein concentration relative to DNA concentration. When a biotin–streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion, allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the full-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin–streptavidin block. We propose a noncanonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein–protein interactions to melt the substrate.
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发表时间：
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