The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities
The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities
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DOI：
10.1093/nar/gkm1025
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发表时间：
2008-02-01
影响因子：
14.9
通讯作者：
Falkenberg, Maria
中科院分区：
文献类型：
--
作者：
Farge, Geraldine;Holmlund, Teresa;Falkenberg, Maria
闪烁蛋白是复制线粒体DNA所需的一种六聚体DNA解旋酶。Flinkle与噬菌体T7基因4蛋白(GP4)有惊人的序列相似性，GP4是噬菌体DNA复制分叉所需的双功能启动酶-解旋酶。人类Flinkle的N-末端结构域包含一些在T7 GP4的N-末端引物结构域中发现的特征序列基序，但其他重要的基序缺失。Flinkle在体外并不是一种活性的启动子酶，N-末端区域的功能仍然难以捉摸。在这份报告中，我们证明了Flinkle的N端部分是有效与单链DNA结合所必需的。该区域的截短降低了DNA解旋酶的活性和线粒体DNA复制体的处理能力。我们还发现，GP4和Flinkle在功能上是不同的。与噬菌体蛋白不同，Flinkle与双链DNA结合。此外，即使在没有镁离子或NTPs的情况下，Flinkle也会形成稳定的六聚体，这表明需要一种辅助蛋白--解旋酶加载器来将Flinkle装载到环状mtDNA基因组上。
The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg2+ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.