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首页> 外文期刊>Nucleic Acids Research >Improved artificial origins for phage phi 29 terminal protein-primed replication. Insights into early replication events
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Improved artificial origins for phage phi 29 terminal protein-primed replication. Insights into early replication events

机译：噬菌体phi 29末端蛋白引发的复制的改良人工起源。洞悉早期复制事件

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摘要

The replication machinery of bacteriophage phi 29 is a paradigm for protein-primed replication and it holds great potential for applied purposes. To better understand the early replication events and to find improved origins for DNA amplification based on the phi 29 system, we have studied the end-structure of a double-stranded DNA replication origin. We have observed that the strength of the origin is determined by a combination of factors. The strongest origin (30-fold respect to wt) has the sequence CCC at the 3' end of the template strand, AAA at the 5' end of the non-template strand and 6 nucleotides as optimal unpairing at the end of the origin. We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ss-DNA regions have very low activity. Most of the effect of the improved origins takes place at the passage between the terminal protein-primed and the DNA- primed modes of replication by the DNA polymerase suggesting the existence of a thermodynamic barrier at that point. We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.
机译：噬菌体phi 29的复制机制是蛋白质引发的复制的范例，在应用方面具有巨大潜力。为了更好地理解早期复制事件并找到基于phi 29系统的DNA扩增的改进起点，我们研究了双链DNA复制起点的末端结构。我们已经观察到，起源的强度是由多种因素共同决定的。最强的来源（相对于wt而言是30倍）在模板链的3'末端具有序列CCC，在非模板链的5'末端具有AAA序列，并且在起源的末端具有6个核苷酸作为最佳不配对。我们还表明，正确定位的置换链的存在很重要，因为具有5'或3'ss-DNA区域的来源的活性非常低。改善起源的大多数作用发生在DNA聚合酶在末端蛋白引发的复制和DNA引发的复制模式之间的通道上，表明此时存在热力学屏障。我们建议起源和TP / DNA聚合酶复合物的模板和非模板链形成一系列相互作用，这些相互作用控制着末端蛋白质引发的复制的关键开始。

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