Identification of flexible Pif1-DNA interactions and their impacts on enzymatic activities.
Identification of flexible Pif1-DNA interactions and their impacts on enzymatic activities.
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DOI：
10.1093/nar/gkac529
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发表时间：
2022-07-08
影响因子：
14.9
通讯作者：
Lu, Ying
中科院分区：
文献类型：
--
作者：
Li, Jinghua;Ma, Jianbing;Kumar, Vikash;Fu, Hang;Xu, Chunhua;Wang, Shuang;Jia, Qi;Fan, Qinkai;Xi, Xuguang;Li, Ming;Liu, Haiguang;Lu, Ying
生物分子复合物中的柔性区域虽然对理解结构-功能关系至关重要，但在高分辨率晶体结构中往往不清楚。在这项研究中，我们发现单分子技术与计算建模相结合，可以表征高分辨率结构确定方法无法解决的动态构象。以两个Pif1解旋酶（ScPif1和BsPif1）为模型系统，我们发现，除了少数紧密结合的核苷酸外，相邻的溶剂暴露核苷酸与解旋酶表面动态相互作用。整个核苷酸片段具有弯曲的构象，并覆盖了解旋酶的两个reca样结构域，这是寸虫机制所必需的。协同方法表明，暴露的核苷酸与解旋酶之间的相互作用可以通过大的拉伸力或用高浓度盐静电屏蔽来减少，从而导致解旋酶的易位率降低。暴露的核苷酸和解旋酶之间的动态相互作用奠定了其酶活性的力和盐依赖性。目前基于单分子的方法补充了破译解旋酶分子机制的高分辨率结构方法。
Flexible regions in biomolecular complexes, although crucial to understanding structure–function relationships, are often unclear in high-resolution crystal structures. In this study, we showed that single-molecule techniques, in combination with computational modeling, can characterize dynamic conformations not resolved by high-resolution structure determination methods. Taking two Pif1 helicases (ScPif1 and BsPif1) as model systems, we found that, besides a few tightly bound nucleotides, adjacent solvent-exposed nucleotides interact dynamically with the helicase surfaces. The whole nucleotide segment possessed curved conformations and covered the two RecA-like domains of the helicases, which are essential for the inch-worm mechanism. The synergetic approach reveals that the interactions between the exposed nucleotides and the helicases could be reduced by large stretching forces or electrostatically shielded with high-concentration salt, subsequently resulting in reduced translocation rates of the helicases. The dynamic interactions between the exposed nucleotides and the helicases underlay the force- and salt-dependences of their enzymatic activities. The present single-molecule based approach complements high-resolution structural methods in deciphering the molecular mechanisms of the helicases.
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影响因子：
14.9
作者：
George T;Wen Q;Griffiths R;Ganesh A;Meuth M;Sanders CM
通讯作者：
Sanders CM
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影响因子：
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作者：
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作者：
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BsPif1 解旋酶的晶体结构揭示 DNA 解旋需要 2B SH3 结构域的主要运动
影响因子：
14.9
作者：
Chen WF;Dai YX;Duan XL;Liu NN;Shi W;Li N;Li M;Dou SX;Dong YH;Rety S;Xi XG
通讯作者：
Xi XG
影响因子：
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作者：
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通讯作者：
Raney, Kevin D.