TYPE-III RESTRICTION ENDONUCLEASES TRANSLOCATE DNA IN A REACTION DRIVEN BY RECOGNITION SITE-SPECIFIC ATP HYDROLYSIS
TYPE-III RESTRICTION ENDONUCLEASES TRANSLOCATE DNA IN A REACTION DRIVEN BY RECOGNITION SITE-SPECIFIC ATP HYDROLYSIS
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DOI：
10.1002/j.1460-2075.1995.tb07296.x
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发表时间：
1995-06-15
期刊：
影响因子：
11.4
通讯作者：
SCHROEDER, C
中科院分区：
文献类型：
--
作者：
MEISEL, A;MACKELDANZ, P;SCHROEDER, C
III 型限制/修饰系统识别短的非回文序列，其中只有一条链可以甲基化，III 型修饰 DNA 的复制产生完全未甲基化的识别位点，根据经典的限制机制，这些识别位点应该是限制信号。我们之前已经表明，如果所有未修饰位点都处于相同方向，则可以防止 III 型酶 EcoP15I 的自杀性限制：EcoP15I 的限制需要一对未甲基化的反向识别位点，我们现在已经解决了位点定向特异性 DNA 限制的分子机制。 EcoP15I 被证明具有内在的 ATP 酶活性，即 DNA 易位的潜在驱动力，ATP 酶活性是唯一识别位点特异性的，但 EcoP15I 修饰的位点也支持该反应，EcoP15I DNA 限制模式由酶与位点的比率预先确定，因为位点饱和酶水平仅在最接近的一对头对头定向位点之间引发切割，DNA 限制被 Lac 阻遏物结合阻断在两个 EcoP15I 位点之间的插入序列中。这些结果排除了 DNA 环，并强烈表明切割是由两个聚合跟踪 EcoP15I-DNA 复合物的紧密接近所触发的。
Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated, Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction, We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites, We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation, The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction, EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites, DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.