Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease
Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease
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DOI：
10.1093/nar/29.14.3069
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发表时间：
2001-07-15
影响因子：
14.9
通讯作者：
Seo, YS
中科院分区：
文献类型：
--
作者：
Bae, SH;Kim, JA;Seo, YS
为了深入了解结构基础！为了了解参与冈崎片段加工的多功能 Dna2 酶，我们进行了生化、生物物理和遗传学研究来剖析 Dna2 的结构域结构。使用枯草杆菌蛋白酶对 Dna2 进行蛋白水解消化产生了 127 kDa 的多肽，该多肽缺乏 Dna2 的 45 kDa N 末端区域。进一步消化产生两个大小大致相等的抗枯草杆菌蛋白酶核心片段，分别为 58 和 60 kDa。令人惊讶的是，与完整酶相比，消化导致 ATP 酶和核酸内切酶活性显着增加（3 至 8 倍）。然而，具有缺乏相应 N 末端区域的突变 DNA2 等位基因的细胞生长严重受损，无法在 37 摄氏度下生长，这表明 N 末端区域包含对 Dna2 细胞功能至关重要的结构域。对缺乏 N 末端 45 kDa 结构域的野生型和/或突变型 Dna2 的流体动力学特性和体内复合物形成的分析表明，Dna2 作为单体具有活性，因此突变型 Dna2 蛋白的缺陷不是由于其无法多聚化。此外，我们发现 N 端 45 kDa 结构域与位于两个催化结构域之间的中心区域发生物理相互作用。我们的结果表明，Dna2 的 N 端 45 kDa 结构域在 Dna2 酶活性的调节中发挥着关键作用，作为 Dna2 最佳功能所必需的分子内和分子间相互作用位点：在冈崎片段加工中。基于我们最近的发现，复制蛋白 A 在冈崎片段加工过程中与 Dna2 发生功能上和物理上的相互作用，讨论了 Dna2 可能的调控模式。
In order to gain insights into the structural basis! of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtillsin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degreesC, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2: in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.