The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity
The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity
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DOI：
10.1073/pnas.97.14.7721
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
Lanka, E
中科院分区：
文献类型：
--
作者：
Deneke, J;Ziegelin, G;Lanka, E
大肠杆菌噬菌体N15编码一种微酸性、630个残基、分子量为72.2 kDa的蛋白质，称为原核端粒酶（TelN）。TelN是N15复制系统的一个组成部分，被认为参与线性原噬菌体DNA的产生。这种线性DNA分子具有共价闭合的末端。在体外催化将环状质粒转化为线性分子的反应。我们证明了在没有其他N15编码因子的情况下，telN的产物起原核端粒酶的作用。纯化的TelN处理含有所提出的靶位点telRL的环状和线性质粒DNA，以产生具有共价闭合末端的线性双链DNA。56-bp的telRL靶位点由22 bp的中心telO回文序列和两个包含反向重复的14-bp侧翼序列组成。telO与这些重复序列在每一侧相隔3bp。telRL序列足以用于TelN介导的加工。体外产生的DNA分子的末端具有与体内观察到的相同的构型。TelN在协同作用中发挥其作为切割-连接酶的活性。
Escherichia coli phage N15 encodes the slightly acidic, 630-residue protein of 72.2 kDa called protelomerase (TelN). TelN is a component of the N15 replication system proposed to be involved in the generation of the linear prophage DNA. This linear DNA molecule has covalently closed ends. The reaction converting circular plasmids into linear molecules was catalyzed in vitro. We demonstrate that the product of telN functions as the protelomerase in the absence of other N15-encoded factors. Purified TelN processes circular and linear plasmid DNA containing the proposed target site telRL to produce linear double-stranded DNA with covalently closed ends. The 56-bp telRL target site consists of a central telO palindrome of 22 bp and two 14-bp flanking sequences comprising inverted repeats. telO is separated from these repeats by 3 bp on each side. The telRL sequence is sufficient for TelN-mediated processing. The ends of the DNA molecules generated in vitro have the same configuration as do those observed in vivo. TelN exerts its activity as cleaving-joining enzyme in a concerted action.