A comparison of eubacterial and archaeal structure-specific 5′-exonucleases
A comparison of eubacterial and archaeal structure-specific 5′-exonucleases
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DOI：
10.1074/jbc.274.30.21387
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发表时间：
1999-07-23
影响因子：
4.8
通讯作者：
Lyamichev, VI
中科院分区：
文献类型：
--
作者：
Kaiser, MW;Lyamicheva, N;Lyamichev, VI
真核菌的DNA聚合酶I蛋白的8′-外切酶结构域与真核菌和古菌的FEN1蛋白是一个具有结构特异性的5′-外切酶家族的成员，它们具有相似的功能，但序列相似性有限。它们的生理作用是在置换合成过程中去除DNA聚合酶产生的移位的5‘链，从而为DNA连接酶创造底物。本文定义了水热菌、嗜热热菌、富氏古舌菌、富氏焦球菌、jannaschii甲烷球菌和热自养甲烷菌对5’-外切酶酶的底物需求。这些酶的最佳底物类似于正在进行链置换合成的DNA，由分叉的下游双工和直接毗邻的上游双工组成，上游双工与下游双工重叠一个碱基对。重叠的单一碱基导致酶在切割后留下缺口，并且比没有重叠的底物切割速度快几个数量级。下游的双链需要10个碱基对或更长的长度，大多数酶才能有效切割。对于大多数酶来说，上游双链只需要2或3个碱基对，并且似乎与引物链的最后一个碱基相互作用。总的来说，这些酶显示出非常相似的底物特异性，尽管它们的序列相似性有限。
The 8'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase, In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.