Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.
Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.
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DOI：
10.1371/journal.pbio.0050020
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发表时间：
2007-02
期刊：
影响因子：
9.8
通讯作者：
Vindigni, Alessandro
中科院分区：
文献类型：
--
作者：
Muzzolini, Laura;Beuron, Fabienne;Patwardhan, Ardan;Popuri, Venkateswarlu;Cui, Sheng;Niccolini, Benedetta;Rappas, Mathieu;Freemont, Paul S;Vindigni, Alessandro
RECQ解旋酶是维持染色体稳定性所必需的。除了DNA解旋外，一些RecQ酶还具有内在的DNA链退火活性。然而，这种双重酶活性的功能和调节机制尚不清楚。在这里，我们描述了人类RECQ1解旋酶的两种四元形式，与五聚体或六聚体一致的高阶低聚物，以及与单体或二聚体一致的较小的低聚物。尺寸排斥层析和透射电子显微镜显示，两种组装态之间的平衡受到单链DNA和三磷酸腺苷结合的影响，三磷酸腺苷或三磷酸腺苷γS倾向于较小的低聚形式。我们对人类RECQ1进行的三维电子显微镜重建显示了一个大约120？×130？的复杂笼状结构，中间有一个孔。这种低聚体结构在RECQ1擅长链退火的条件下稳定下来。相比之下，与ATPase缺失的K119R和E220Q突变体的竞争实验表明，DNA解离需要RECQ1单体或紧密结合的二聚体。总而言之，我们的发现表明，高阶低聚物与DNA链退火有关，而低阶低聚物与DNA解离有关。DNA双螺旋的瞬间打开是DNA代谢过程中的一个基本步骤。这个反应是由称为解旋酶的蛋白质驱动的，解旋酶利用三磷酸腺苷作为燃料来解开DNA双链。RecQ解旋酶家族有助于维持基因组的稳定性。最近的研究表明，RecQ解旋酶除了促进DNA解离外，还可以催化相反的反应-部分解离的DNA双链的配对。然而，调节这种双重酶活性的潜在机制尚不清楚。在这里，我们描述了人类RECQ1解旋酶的两种结构形式，一种是由五个或六个亚基组成的大的寡聚复合体，另一种是仅与一个或两个分子一致的较小的形式。我们提供了一个较大的复合体的三维结构的初步视图，并表明这种状态与DNA链退火有关，而较小的形式进行DNA解旋。从链退火到DNA解离的功能切换是由ATP结合控制的，这促进了更大、更高级别的复合体的解离。通过提供对调节RecQ解旋酶活性的机制的洞察，我们的研究打开了了解DNA代谢的一个基本方面的新窗口。RecQ解旋酶可以解开或退火链的DNA。这种双重功能可以通过电子显微镜研究来解释；高阶低聚物似乎负责退火，而低阶低聚物控制DNA的解旋。
RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding. The transient opening of the DNA double helix is a fundamental step in several DNA metabolic processes. This reaction is driven by proteins called helicases, which make use of ATP as fuel to unwind the DNA duplex. The RecQ family of helicases help maintain genome stability. Recent studies have shown that RecQ helicases, in addition to promoting DNA unwinding, can also catalyze the opposite reaction—the pairing of the partially unwound DNA duplexes. The mechanisms underlying the regulation of this dual enzymatic activity are, however, unknown. Here we describe two structural forms of the human RECQ1 helicase, a large oligomeric complex composed of five or six subunits, and a smaller form consistent with only one or two molecules. We provide an initial view of the three-dimensional structure of the larger complex and show that this state is associated with DNA strand annealing, whereas the smaller form carries out DNA unwinding. The functional switch from strand-annealing to DNA unwinding is controlled by ATP binding, which promotes the dissociation of the larger, higher-order complexes. By providing insight into the mechanisms regulating RecQ helicase activity, our study opens a new window onto a fundamental aspect of DNA metabolism. RecQ helicases can either unwind or anneal strands of DNA. This dual functionality is explained by electron microscopy studies; higher-order oligomers appear responsible for annealing, whereas lower-order oligomers control DNA unwinding.