Functional uncoupling of twin polymerases - Mechanism of polymerase dissociation from a lagging-strand block
Functional uncoupling of twin polymerases - Mechanism of polymerase dissociation from a lagging-strand block
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DOI：
10.1074/jbc.m401649200
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发表时间：
2004-05-14
影响因子：
4.8
通讯作者：
O'Donnell, M
中科院分区：
文献类型：
--
作者：
McInerney, P;O'Donnell, M
复制分叉经常会受到导致分叉停滞、停止或崩溃的事件的影响。使用合成的滚环DNA底物，我们证明了对滞后链聚合酶的阻断不会影响解旋酶或前导链聚合酶的活性。事实上，滞后链合成也在继续。因此，被阻断的滞后链酶迅速从阻断位点解离，并在新的启动位点上恢复合成。此外，滞后的聚合酶被持续阻断的研究表明，即使领先的聚合酶仍然附着在滞后的链酶上，它的作用也没有减弱。因此，在遇到滞后支架的阻挡块时，聚合酶在功能上解偶联但在物理上保持联系。进一步的研究表明，裸露的单链DNA会导致从其β-DNA底物上破坏停滞不前的聚合酶。因此，随着复制体的进展，积聚在滞后链模板上的单链DNA环在SSB蛋白耗尽后从β释放停滞的滞后链聚合酶。然后，滞后链聚合酶可以自由地继续生产冈崎片段。
Replication forks are constantly subjected to events that lead to fork stalling, stopping, or collapse. Using a synthetic rolling circle DNA substrate, we demonstrate that a block to the lagging-strand polymerase does not compromise helicase or leading-strand polymerase activity. In fact, lagging-strand synthesis also continues. Thus, the blocked lagging-strand enzyme quickly dissociates from the block site and resumes synthesis on new primed sites. Furthermore, studies in which the lagging polymerase is continuously blocked show that the leading polymerase continues unabated even as it remains attached to the lagging-strand enzyme. Hence, upon encounter of a block to the lagging stand, the polymerases functionally uncouple yet remain physically associated. Further study reveals that naked single-stranded DNA results in disruption of a stalled polymerase from its beta-DNA substrate. Thus, as the replisome advances, the single-stranded DNA loop that accumulates on the lagging-strand template releases the stalled lagging-strand polymerase from beta after SSB protein is depleted. The lagging-strand polymerase is then free to continue Okazaki fragment production.