Linear diffusion of restriction endonucleases on DNA.
Linear diffusion of restriction endonucleases on DNA.
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DNA 上限制性核酸内切酶的线性扩散。
DOI：
10.1016/s0021-9258(18)88951-7
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
C. Gualerzi
中科院分区：
文献类型：
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作者：
H. Ehbrecht;A. Pingoud;C. Urbanke;G. Maass;C. Gualerzi
我们研究了 EcoRI、HindIII 和 BamHI 切割 DNA 的速率对底物链长的依赖性。为了保持侧翼序列和非特异性结合的影响对于所有底物相同，我们使用相同的质粒DNA进行了所有实验，该质粒DNA先前已用多种不同的限制性酶消化以给出一组不同长度的底物。我们的结果表明，根据缓冲液条件，长底物比小底物裂解得更快。我们将这些发现解释为，在某些条件下，酶在 DNA 上的线性扩散参与了识别位点的定位。对于 EcoRI，在 1 mM MgCl2 下，平均扩散长度约为 1000 个碱基对，可以通过扩散理论证明其对应于 5 X 10(-10) cm2 s-1 的线性扩散系数。在 10 mM MgCl2 下，EcoRI 的线性扩散可以忽略不计，并且不会导致位点定位率显着提高。在存在非饱和量的原核组蛋白样蛋白 Hu (NS 2) 之一的情况下，EcoRI 以相同的速率切割小 DNA 底物和大 DNA 底物，这表明与 DNA 结合的其他蛋白质构成了无法发生线性扩散的屏障。我们得出的结论是，线性扩散尽管在体外某些条件下可检测到，但对于体内位点定位过程可能并不重要。
We have investigated the dependence of the rate of cleavage of DNA by EcoRI, HindIII, and BamHI on the chain length of the substrate. In order to keep the influence of flanking sequences and of nonspecific binding identical for all substrates we have carried out all experiments with the same plasmid DNA which had been digested previously with a variety of different restriction enzymes to give a set of substrates of different lengths. Our results show that depending on the buffer conditions long substrates are cleaved faster than small ones. We interpret these findings to mean that under certain conditions a linear diffusion of the enzymes on the DNA is involved in localizing the recognition sites. For EcoRI the mean diffusion length is approximately 1000 base pairs at 1 mM MgC12 which can be shown by diffusion theory to correspond to a linear diffusion coefficient of 5 X 10(-10) cm2 s-1. At 10 mM MgCl2 the linear diffusion of EcoRI is negligible and does not lead to a significant enhancement of the rate of site localization. In the presence of nonsaturating amounts of one of the prokaryotic histone-like protein Hu (NS 2) small and large DNA substrate are cleaved with identical rate by EcoRI indicating that other proteins bound to the DNA constitute a barrier across which linear diffusion cannot take place. We conclude that linear diffusion, albeit detectable under certain conditions in vitro, probably is of little importance for the process of site localization in vivo.