Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome
Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome
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DOI：
10.1073/pnas.1604894113
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发表时间：
2016-05-24
影响因子：
11.1
通讯作者：
Richardson, Charles C.
中科院分区：
文献类型：
--
作者：
Hernandez, Alfredo J.;Lee, Seung-Joo;Richardson, Charles C.
由于DNA链的反向平行以及酶促DNA合成的极性，DNA复制是半不连续进行的。尽管前导链是连续合成的，但滞后链是以被称为冈崎片段的小片段形式合成的。滞后链的合成是一个复杂的过程，需要RNA引物合成、转移到滞后链聚合酶以及由DNA引发酶和滞后链聚合酶协同作用进行延伸等重复循环。我们利用噬菌体T7复制系统研究了控制冈崎片段起始的事件。T7 DNA聚合酶利用引物的速度比引物形成的速度慢。DNA引发酶缓慢释放引物使得聚合酶能够与复合物结合，随后是一个缓慢的引物交接步骤。T7单链DNA结合蛋白提高了引物形成和延伸效率，但促进的引物延伸轮次有限。我们提出了一个模型，描述冈崎片段的起始、片段长度的调控以及它们对协调的前导链和滞后链DNA合成的影响。
DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.