DNA AND SPERMIDINE PROVIDE A SWITCH MECHANISM TO REGULATE THE ACTIVITY OF RESTRICTION ENZYME NAE-I
DNA AND SPERMIDINE PROVIDE A SWITCH MECHANISM TO REGULATE THE ACTIVITY OF RESTRICTION ENZYME NAE-I
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DOI：
10.1073/pnas.86.24.9707
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
TOPAL, MD
中科院分区：
文献类型：
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作者：
CONRAD, M;TOPAL, MD
序列特异性DNA-蛋白质相互作用是DNA功能的基础。为了更好地理解这些相互作用，我们研究了位置对II型限制性内切酶（EC 3.1.21.4）Nae I切割DNA的影响。我们发现了两类Nae I限制性位点：敏感位点和耐切割位点。动力学分析表明，Nae I被含有可裂解Nae I位点的DNA激活，通过非竞争性机制快速裂解耐Nae I位点，底物DNA的Km约为2 nM，激活DNA的KA约为6 nM;激活增加催化作用，但不增加底物结合。体外缺失突变表明，侧翼序列的Nae I识别位点负责激活和非激活Nae I位点之间的差异。多胺亚精胺有一个显着的影响，Nae I与DNA的相互作用，在1 mM的亚精胺的存在下，耐药位点被迅速切割和可切割的DNA抑制切割。反式DNA序列对酶活性的直接调节，以及对细胞周期敏感的多胺的这种调节的调节，提供了一种调节开关机制。这种开关的生物控制功能的影响进行了讨论。
Sequence-specific DNA-protein interactions are basic to DNA function. To better understand these interactions, we studied the effect of position on cleavage of DNA by the type Ii restriction enzyme (EC 3.1.21.4) Nae I. We discovered two classes of Nae I restriction sites: sites susceptible and sites resistant to cleavage. Kinetic analysis showed that Nae I was activated by DNA containing cleavable Nae I sites to rapidly cleave resistant Nae I sites by a noncompetitive mechanism with a Km for substrate DNA of about 2 nM and a KA for activating DNA of about 6 nM; activation increased catalysis but not substrate biding. Deletion mutagenesis in vitro showed that sequences flanking the Nae I recognition site were responsible for the differences between activating and nonactivating Nae I sites. The polyamine spermidine had a dramatic effect on the interaction of Nae I with DNA; in the presence of 1 mM spermidine, resistant sites were cleaved rapidly and cleavable DNA inhibited cleavage. The direct regulation of enzymatic activity by DNA sequences in trans, and the modulation of this regulation of a polyamine that is sensitive to the cell cycle, provides a regulatory switch mechanism. The implications of this switch for biological control functions are discussed.