Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex.
Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex.
复制标题
HCV NS3 解旋酶对核酸的解旋对双链体的结构敏感。
DOI：
10.1093/nar/29.2.565
复制
发表时间：
2001
影响因子：
14.9
通讯作者：
Raney,KD
中科院分区：
文献类型：
--
作者：
Tackett,AJ;Wei,L;Cameron,CE;Raney,KD
丙型肝炎病毒 (HCV) 解旋酶、非结构蛋白 3 (NS3) 被认为有助于 HCV 基因组复制，并被认为是抑制 HCV 的靶标。为了研究 NS3 核酸解旋的底物要求，通过将 30 聚体寡核苷酸退火至 15 聚体来制备底物。所得的 15 bp 双链体包含 15 nt 的单链 DNA 突出端，称为结合链。制备了其他底物，其中 15mer DNA 被肽核酸 (PNA) 链取代。 PNA-DNA 底物被 NS3 解绕，但观察到的链分离速率比同等 DNA-DNA 底物至少慢 25 倍。 NS3 与 PNA-DNA 底物的结合与 DNA-DNA 底物相似，因为 NS3 最初与单链突出端结合，每个底物中的单链突出端都是相同的。在类似条件下，NS3 不会解开 PNA-RNA 底物。相比之下，NS3 对吗啉代-DNA 和硫代磷酸酯-DNA 底物的利用效率与 DNA-DNA 底物相同。这些结果表明，PNA-DNA 和 PNA-RNA 异源双链体采用了不利于 NS3 解旋的结构，表明 NS3 的解旋活性对双链体的结构敏感。
Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.