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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork
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Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

机译：位置特异性DNA碱基对的呼吸波动与调节解旋酶向复制叉的移动有关

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摘要

We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectro-scopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.
机译：我们以前曾使用过近紫外圆二色性和DNA碱基类似物探针放置位点的荧光光谱的变化，专门显示模型复制叉子构建物中叉子连接处的前三个碱基对通过生理条件下的“呼吸”波动而显着打开。在这里，我们使用这些探针提供DNA叉与溶液中紧密结合的复制解旋酶的初始相互作用的机制快照。噬菌体T4的primosome解旋酶由六个（gp41）解旋酶亚基，一个（gp61）primase亚基和不可水解的GTPγS组装而成。当与DNA复制叉构建体结合时，该复合物使一个碱基对进入叉的双链体部分，并形成稳定结合的解旋酶“起始复合物”。用GTP替代GTPγS可以完成解旋酶驱动的退绕过程。我们的光谱探针显示，这种稳定的解旋酶起始复合物中的primosome主要通过骨架接触结合叉的DNA，并使叉的第一互补碱基对保持开放构象，而第二，第三和第四碱基对双链体的“α”基本上显示了先前表现为自由叉的前三个碱基对的呼吸行为。这些光谱变化以及动态荧光猝灭结果与原始体结合模型一致，在该模型中，滞后的DNA链穿过六角解旋酶的中心孔，前导链与解旋酶亚基的“外部”表面结合六聚体，并且单个引发酶亚基与两条链相互作用。

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