Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm.
Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm.
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DOI：
10.1186/1472-6807-9-2
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发表时间：
2009-01-22
影响因子：
--
通讯作者：
Morikawa K
中科院分区：
文献类型：
--
作者：
Oyama T;Oka H;Mayanagi K;Shirai T;Matoba K;Fujikane R;Ishino Y;Morikawa K
高温焦球菌Hjm （PfuHjm）是一种结构特异性DNA解旋酶，最初是通过体外筛选Holliday结迁移活性发现的。它属于解旋酶超家族2，与参与DNA修复的人类DNA聚合酶Θ （PolΘ）、HEL308和果蝇Mus308蛋白具有同源性。先前的生化和遗传分析表明，PfuHjm优先结合叉相关的y结构dna并解绕其双链区域，这表明该解旋酶是细菌RecQ解旋酶的功能对偶物，对基因组的维持至关重要。通过PfuHjm阐明DNA解绕和易位机制将需要其在原子分辨率上的三维结构。我们确定了PfuHjm的晶体结构，两种载子态和两种核苷酸结合形式，分辨率为2.0-2.7 Å。无论核苷酸结合状态如何，核苷酸结合位点周围的整体结构和局部构象几乎相同，包括侧链构象。Hjm的结构与fulgidus Archaeoglobus Hel308的结构相似。通过将Hjm的5个结构域拟合到对应的Hel308结构域上，构建了Hjm-DNA复合物模型，结果表明Hjm与DNA的相互作用与Hel308相似。值得注意的是，与jhm结合的硫酸盐离子位于假定的DNA结合表面上。hhm -DNA复合物的电镜分析显示，DNA双链区域具有相当大的柔韧性，可能是由于蛋白质-DNA相互作用特别弱。我们目前的结构可以合理地建立人类PolΘ解旋酶区域的同源性模型，表明古细菌和真核生物之间具有很强的构象保守性。我们的无DNA PfuHjm结构与Hel308的DNA复合物结构的详细比较表明，相似的DNA解绕和易位机制，这可以推广到同一家族的所有成员。结构比较也暗示了在DNA解绕反应中5个结构域的轻微重排。DNA双链区域和酶之间意想不到的小接触似乎有利于过程解旋酶的活性。
Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Θ (PolΘ), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution. We determined the crystal structures of PfuHjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0–2.7 Å. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of Archaeoglobus fulgidus Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolΘ, indicating the strong conformational conservation between archaea and eukarya. The detailed comparison between our DNA-free PfuHjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.
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