Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase.
Unwinding forward and sliding back: an intermittent unwinding mode of the BLM helicase.
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向前解卷和向后滑动：BLM解旋酶的间歇解卷模式
DOI：
10.1093/nar/gkv209
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发表时间：
2015-04-20
影响因子：
14.9
通讯作者：
Li M
中科院分区：
文献类型：
--
作者：
Wang S;Qin W;Li JH;Lu Y;Lu KY;Nong DG;Dou SX;Xu CH;Xi XG;Li M
有证据表明，布卢姆综合征解旋酶，BLM，催化停滞的复制叉的回归和破坏在同源重组（HR）过程中形成的位移环（D-环）。在这里，我们构建了一个3′单链缺口和5′双链手柄的叉状DNA，以部分模拟停滞的DNA叉，并使用磁镊研究BLM催化的叉状DNA解旋。我们已经直接观察到，BLM解旋酶可以在不同的力下双链体解旋后在相对链上滑动一段距离。对于具有长发夹的DNA构建体，发夹的渐进解旋经常被酶的链转换和向后滑动中断。定量研究的不间断的解绕长度（时间）揭示了一个两态转换机制的链开关在解绕过程中。突变研究表明，RQC结构域在稳定解旋酶/DNA相互作用过程中起着重要的作用，DNA解旋和BLM的向后滑动。特别是，在RQC结构域中的Lys 1125，在RecQ解旋酶中高度保守的氨基酸，可能参与向后滑动活性。我们还直接观察了BLM破坏模拟停滞复制叉的体外途径。这些结果可能为BLM在DNA修复和同源重组中的作用机制提供新的线索。
There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination.
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影响因子：
16.6
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
Marians, KJ
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作者：
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通讯作者：
Hickson, Ian D.