The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities
The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities
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DOI：
10.1074/jbc.m110131200
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发表时间：
2002-02-15
影响因子：
4.8
通讯作者：
Slesarev, AI
中科院分区：
文献类型：
--
作者：
Belova, GI;Prasad, R;Slesarev, AI
拓扑异构酶 V (Topo V) 是 IB 型（类真核）DNA 拓扑异构酶。它是在超嗜热原核生物 Methanopyrus kandleri 中发现的，是唯一具有相关无嘌呤/无嘧啶 (AP) 位点加工活性的拓扑异构酶。通过 37°C 和 80°C 下的有限蛋白水解来探测游离状态和 DNA 结合状态的 Topo V 的结构。 Topo V 蛋白由以下部分组成：(i) 44-kDa NH2 末端核心子结构域，其中包含拓扑异构酶活性的活性位点酪氨酸残基；(ii) 紧邻的 16-kDa 子结构域，包含简并螺旋-发夹-螺旋 (HhH) 基序；(iii) 蛋白酶敏感的 18-kDa HhH“铰链”区域；以及 (iv) 34-kDa COOH 末端 HhH 结构域。克隆、纯化并表征了包含 NH2 末端 44-kDa 和 16-kDa 结构域 (Topo61)、44-、16-和 18-kDa 结构域 (Topo78) 以及 COOH 末端 34-kDa 结构域 (Topo34) 的三种截短的 Topo V 多肽。 Topo61 和 Topo78 都是活性拓扑异构酶，但与 Topo V 不同的是，这些酶会受到高盐浓度的抑制。 Topo34具有很强的DNA结合能力，但没有拓扑异构酶活性。最后，我们证明 Topo78 和 Topo34 具有 AP 裂解酶活性，这在碱基切除 DNA 修复中很重要。因此，Topo V 具有至少两个能够处理 AP DNA 的活性位点。讨论了多个 HhH 基序对 Topo V 持续合成能力的重要性。
Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degreesC and 80 degreesC. The Topo V protein is comprised of (i) a 44-kDa NH2-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH2-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.