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首页> 外文期刊>Nucleic Acids Research >Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro.
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Subunit assembly modulates the activities of the Type III restriction-modification enzyme PstII in vitro.

机译：亚基组装在体外调节III型限制性修饰酶PstII的活性。

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摘要

We demonstrate that, like other Type III restriction endonuclease, PstII does not turnover such that a DNA substrate is only fully cleaved at a Res2Mod2-to-site ratio of approximately 1:1. However, unlike other Type III enzymes, the cleavage rate profiles varied with protein concentration: using 5 nM DNA and 25 nM PstII, approximately half of the DNA was cut at a fast rate while the remainder was cut 24 times more slowly; in comparison, with 100 nM PstII cleavage occurs at a single fast rate. The inclusion of the methyl donor S-adenosyl methionine does not alter the rates with 100 nM PstII but with 25 nM PstII the reaction stopped after completion of the initial fast cleavage phase owing to methylation. Concentration-dependent rates were also observed in methylation assays: at 100 nM PstII, a single slow rate was measured while at lower PstII concentrations both fast and slow rates were measured. We propose a model in which the intact Res2Mod2 complex favoured at high PstII concentrations is a fast endonuclease/slow methyltransferase while the various subassemblies which coexist at lower concentrations are fast methyltransferases. A potential role for disassembly in control of restriction activity in vivo is discussed.
机译：我们证明，像其他III型限制性核酸内切酶一样，PstII不更替，因此DNA底物仅在Res2Mod2与位点比例大约为1：1时被完全切割。但是，与其他III型酶不同，切割速率曲线随蛋白质浓度的变化而变化：使用5 nM DNA和25 nM PstII，大约一半的DNA被快速切割，而其余的则被缓慢切割24倍。相比之下，以100 nM的PstII裂解速度是单一的。甲基供体S-腺苷甲硫氨酸的加入不会改变100 nM PstII的反应速率，但是对于25 nM PstII，由于甲基化，反应在初始快速裂解阶段完成后就停止了。在甲基化分析中还观察到浓度依赖性的速率：在100 nM PstII下，测量了一个慢速，而在较低的PstII浓度下，测量了快速和慢速。我们提出了一个模型，其中完整的Res2Mod2复合物在高PstII浓度下受青睐是快速核酸内切酶/慢甲基转移酶，而在较低浓度下共存的各种子组件是快速甲基转移酶。讨论了在控制体内限制活性中拆卸的潜在作用。

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