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首页> 外文期刊>Biochemistry >Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I.
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Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I.

机译：末端碱基对氢键在DNA聚合酶I的Klenow片段的3'端校对中的重要性。

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摘要

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymerase I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mimics of thymine and adenine, respectively, are used to investigate the effects of base pair geometry and stability on the rate of this exonuclease activity. Steady-state kinetics measurements show that complementary T.A base pairs at the end of a primer-template duplex are edited 14-40-fold more slowly than mismatches. By contrast, a 3'-end T residue in a T. Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact that it resembles a T.A pair in structure. Similarly, the A in an A.F pair is edited as rapidly as a mismatched pair despite its close structural mimicry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicating specific interactions between the exonuclease domain and the base of the nucleotide being edited. Finally, thermal denaturation studies are carried out to investigate the relationship between editing and the ease of unwinding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these base pairs when placed in a context resembling a primer-template duplex. In general, the rate of 3'-end editing appears to be governed by the rate of fraying of the DNA terminal pair, and base pair geometry appears to have little effect.
机译：我们描述旨在评估由大肠杆菌DNA聚合酶I的Klenow片段控制3'-末端校对速度的物理因素的研究。两种非极性脱氧核苷等位基因，分别含有2,4-二氟甲苯（F）和4-甲基苯并咪唑（Z）分别是胸腺嘧啶和腺嘌呤的非氢键结合形状模拟物，用于研究碱基对的几何形状和稳定性对该核酸外切酶活性速率的影响。稳态动力学测量表明，引物-模板双链体末端的互补T.A碱基对编辑比错配慢14-40倍。相比之下，T.Z对中的3'端T残基以与天然碱基错配相当的速率进行编辑，尽管它在结构上类似于T.A对。类似地，尽管A.F对中的A结构紧密模仿A.T对，但是A.F对中的A与错配对一样快地被编辑。有趣的是，当碱基对反向且F或Z位于3'末端时，它们的编辑速度会更慢，可能暗示核酸外切酶结构域与被编辑核苷酸的碱基之间存在特定的相互作用。最后，进行热变性研究以研究双链体的编辑和展开容易程度之间的关系。当置于类似于引物-模板双链体的环境中时，在双链体末端对F或Z残基相反的碱基的快速编辑似乎与这些碱基对的稳定性很好地相关。通常，3'端编辑的速率似乎受DNA末端对磨损的速率控制，而碱基对的几何形状似乎影响很小。

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