The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis.
The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis.
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DOI：
10.1016/j.jmb.2011.09.039
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发表时间：
2011-11-18
影响因子：
5.6
通讯作者：
McHenry, Charles S.
中科院分区：
文献类型：
--
作者：
Dohrmann, Paul R.;Manhart, Carol M.;Downey, Christopher D.;McHenry, Charles S.
冈崎片段合成完成后，滞后链复制酶必须在0.1秒内循环到复制叉处的下一个引物，以维持DNA合成的生理速率。我们测试了碰撞模型，该模型假定循环是由聚合酶遇到前面冈崎片段的5′-末端触发的。探测与表面等离子体共振，DNA聚合酶III全酶引发复合物上形成固定的缺口模板。引发复合物表现出约15分钟的解离半衰期。将缺口大小减小到一个核苷酸使解离速率增加2.5倍，并且差距的完全填充使解离速率增加另外的3倍（t1/2 ± 2分钟）。在需要完全填充差距的反应中，外源性引发模板和ATP加速解离另外四倍。聚合酶下游的5′-三磷酸或5′-RNA终止的寡核苷酸都不能进一步加速解离。因此，在缺口完成和与下游冈崎片段碰撞时聚合酶释放的速率慢1000倍，以至于不能支持足够的循环速率，并且可能提供了备用机制以在其他循环信号不存在时使聚合酶释放。动力学测量表明，添加最后一个核苷酸以填充差距不是聚合酶释放和循环的限速步骤。在模型Okazaki片段之间的差距被填充后，观察到适度（约7个核苷酸）的链置换。为了确定感测间隙填充以调节复制酶对模板的亲和力的蛋白质的身份，我们用高反应性和非化学选择性的二氮杂环丙烷进行了光交联实验。只有α亚基交联，表明它作为传感器。
Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 second to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 minutes. Reduction of gap size to one nucleotide increased the rate of dissociation 2.5-fold and complete filling of the gap increased the off rate an additional three-fold (t½ ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional four-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nucleotide) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-crosslinking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating it serves as the sensor.
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DOI：
10.1073/pnas.90.22.10881
发表时间：
1993-11-15
影响因子：
11.1
作者：
KABOORD, BF;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ
影响因子：
64.8
作者：
Lee, JB;Hite, RK;van Oijen, AM
通讯作者：
van Oijen, AM
影响因子：
5.6
作者：
Dohrmann, PR;McHenry, CS
通讯作者：
McHenry, CS
影响因子：
4.8
作者：
Li, XJ;Marians, KJ
通讯作者：
Marians, KJ
影响因子：
64.5
作者：
Lamers, Meindert H.;Georgescu, Roxana E.;Kuriyan, John
通讯作者：
Kuriyan, John