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首页> 外文期刊>Biochemistry >Distinct Facilitated Diffusion Mechanisms by E. coli Type II Restriction Endonucleases
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Distinct Facilitated Diffusion Mechanisms by E. coli Type II Restriction Endonucleases

机译：大肠杆菌II型限制性内切核酸酶的独特促进扩散机制

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The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase, 5'-GAATTC-3'; EcoRV ENase, 5'-GATATC-3'), leaving overhangs and blunt DNA segments, respectively. Here, we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to similar to 600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase, The mechanism displayed by EcoRI ENaseresults in a highly thorough search of DNA, whereas the EcoRV ENase mechanism results in an extended, yet less rigorous, interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the 10-fold higher in vivo concentrations of EcoRI ENase compared to that of EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal that bacteria employ against foreign DNA involves seemingly similar enzymes that rely on distinct but complementary search mechanisms. Our comparative approach reveals bow different proteins utilize distinct site-locating strategies.
机译：蛋白质被动搜索涉及非特异性DNA的特定DNA序列对于基因调节，DNA修复，噬菌体防御以及多种表观遗传过程至关重要。不同的机制促进了这些搜索，关于哪种机制或机制的混合物最适合特定蛋白质，更重要的是其生物学作用，仍未解决。为了解决这个问题，我们比较了两种经过充分研究的细菌限制性核酸内切酶（ENase），EcoRI和EcoRV的转运特性。这些二聚体，镁依赖性酶水解相关位点（EcoRI ENase，5'-GAATTC-3'; EcoRV ENase，5'-GATATC-3'），分别留下突出端和钝的DNA片段。在这里，我们证明了EcoRI ENase的广泛滑动，包括从DNA分离之前滑升至600 bp左右，这与EcoRV ENase对跳变机制的依赖性更大有关，EcoRI ENase显示的机制导致了对DNA的彻底搜索，而EcoRV ENase机制导致对DNA序列空间的扩展但不那么严格的询问。我们描述了如何通过这些内切核酸酶的其他方面来补充这些机理上的区别，例如与EcoRV ENase相比体内浓度高出10倍的EcoRI ENase。此外，我们假设细菌针对外源DNA使用的高度多样化的酶库涉及看似相似的酶，这些酶依赖于独特但互补的搜索机制。我们的比较方法表明弓形不同的蛋白质利用不同的位点定位策略。

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